Lab microbiology

Helpfulness: 0
Set Details Share
created 5 years ago by raheen913
148 views
updated 3 years ago by raheen913
show moreless
Page to share:
Embed this setcancel
COPY
code changes based on your size selection
Size:
X
Show:
1

type of microscope we use

Compound light

2

types of eukaryotes
what type of lens required

mold, pennicillin,, protozoa, plantae, animalia, fungi
10X or 40X

3

types o prokaryotes
what types of lens required

bacteria, archaea
oil immersion 100X

4

types of fungi

aspergillus, penicillium, schizzos/saccoromyces pombeĆ°

5

types of algae

diatons

6

types of protozoa

euglena, paramecium, giardia lamblia

7

types of bacteria

E.coli, staphylococcus aureus

8

Electron microscope is used for?

better resolution or better sharpness ex: 1000X and for smaller wave lenghts. structural details of bacterial cells are viewed with this..

9

disadvantage of electron microscope

kills bacteria due to high beam of electrons, and harsh staining process

10

types of electron microscopes

transmission electron microscope
scanning electron microscope

11

types of modern light microscope

bright field
dark field
phase contrast
flourescent

12

dark field microscope used for

glow in dark, detection of objects that are extremely small.. specimens appear bright against a dark background EX: bacterial flagella

13

phase contrast microscope used for

not able to kill bacteria/ specimen is illuminated with a ring of light

14

scanning electron microscope

for outside view

15

transmission electron microscope

for sections, inside

16

simple wet mount

samples are placed on slides and covered w/coverslip and observed with dark field or phase contrast

17

depression slide mount

use depression slide and suspend a drop of liquid, then cover w/coverslip . offers less restriction to the movement of organisms

18

advantages of staining

increase contrast
stains can be very specific so identifies specific structures of a microorganism
looks prettier/better

19

disadvantages of staining

can kill microorganism
may introduce artefacts
loss of mobility (cannot move)

20

2 methods of observing Unstained samples are:

hanging drop (depression slide)
and
wet mound slide

21

what are simple stains used for

to check if there is bacteria

22

3 types of simple stains are

acidic stains, basic stains, neutral stain

23

acidic stain is what type of stain and carries what charge

anionic
negative (-) charge

24

basic stain is what type of stain and carries what charge

cationic
positive (+) charge

25

give example of neutral stain

india ink

26

give example of acidic stains

congo red
nigrosin
picric acid
safranin??

27

give example of basic stains

crystal violet
methylene blue
carbdfushin
basic fushin
malichle green

28

which stain is most commonly used

basic stain because it attracts the negatively charged bacterial cell towards the postiive basic stain

29

what are differential stain

they are highly specific
they check more than just bacteria

30

give examples of other differential stains

gram stain, spore stain, capsule stain, acid fast stain, flagella stain

31

characteristic of bacteria

they are distinguished by shape

32

shapes of bacteria

bacillus/bacilli (rods)
coccus/cocci (spheral)
spirillum/spirilla (spiral)

33

hanging drop slide is what type of slide

depression slide

34

how to tell if bacterial growth has formed when testing in testtube

gets cloudy
is not translucent

35

as magnification increases, working distance will?

decrease

36

as magnification increases, (NA) numerical aperture will?

increase

37

as magnification increases, resolution will?

increase

38

what is 10X lens power called and what is its NA (numerical aperture)and color

low power lens
NA 0.25 (yellow)

39

what is 40X lens power called,what is its NA and color

high power or high dry lens
NA 0.65 (blue)

40

what is 100X lens power called, what is its NA and color

oil immersion lens
NA 1.25 (white)

41

what is 4X power called, what is its NA and color

scanning lens
NA 0.10 (red)

42

Parfoca

ability to change objective without having to refocus microscope

43

iris diaphragm

located near condensor, light intensity is adjusted by opening or closing the iris diaphragm

44

condensor

light is focused on the speciment by the condensor, situated between light source and specimen

45

WET MOUND SLIDE method

hold slide by edge
stir beaker of pond water
put 1 drop on slide
cover with cover slide
see under low light on microscope (40x)

46

DEPRESSION SLIDE METHOD

take cover slip, use toothpick to apply vaseline to all 4 corners add 1 droplet of pond water, flip depression slide onto cover slip, flip again quickly, view under microscope

47

SIMPLE STAINING METHOD

take clean slide, flame inculating loop and cool off, open testtube of bacteria sample, pass on flame, make smear on clean slide, air dry, take slide and pass over flame, apply stain (meth blue) let it sit for a min. wash excess stain , blot dry, use oil immersion to see on microscope

48

diagphram

controls the amount of lightwhich pases the specimen and drasticaly affects the focus of the image

49

ocular lens

10X maginification, maginifies image formed by objective lens, does not improve resolution, has a pointer

50

arm

connects to base for support and easy to carry

51

light intensity knob

controls the intenstiy of light coming from bulb

52

fine focus adjustment knob

precise focusing, high magnification, high power, oil immersion

53

coarse focus adjustment knob

rapid focusing, scanning lens and low power lens. roughly focuses the object before bringing to fine adjustments

54

stage

holds the slide

55

base

bottom of microscope for support

56

condensor lens

connects to iris diagphram and located under stage. directs light from the lamp through the slide specimen

57

stage clip

fix slide into position

58

types of objective lens

4X scanning
10X low power
40X high power
100X oil immersion

59

types of light microscope

bright field, dark field, flourensence, phase contrast microscopy

60

benefit of using oil immersion

increases resolution and increases numerical aperture of the objective lens

61

numerical aperture

lens abilty to capture light coming from the specimen and use it to make the image

62

difference between light and electron microscope

light M - uses light source, used to watch living cells
Electron M - has beams of electrons focused by magetic lens, used for atoms of heavy metals and used as stains because living tissue is destryoed by the intense beam of electrons.
Major difference in both is Resolution. electron is higher than 1000X.. LIght is upto 100X

63

simple stain are what

method of staining microorganism which uses only one dye

64

negative stain are what

neg stains are simple stains that stains the background and leaves the bacteria unstained

65

types of simple stains

acidic (-)
basic (+)
neutral

66

what are simple stain used for

to find shape, size and arrangements of bacteria

67

what is the purpose of heat fix

so bacteria would adhere to the slide

68

problems that can arise with heat fix

bacteria can get overheated and hence shrink, artifacts

69

best way to heat fix a slide

let it dry completelety before heat fix and dont keep it to close to the flame, move fast..

70

how many stains used in simple and differential stainning

simple - only 1 stain
differential - 2 or more stains

71

purpose of differential staining

identifies specific ones from unknown mixture,
mainly used for research and diagnosis,
distinguishes cell types

72

what is pure culture

a culture that contains a single species of organisms

73

purpose of streak plate

produces isolated colonies of an organism

74

prupose of spread plate

spreads a previously diluted microorganism allowing for colony growth and quantified analysis

75

purpose of pour plate

diluted bacteria is mixed with heated agar and poured into petri, showing the different biochemical needs among the bacteria
To determine the number of microbes/ml or microbes/gram.

76

disadvantage of pour plate

embedded colonies are much smaller than those on surface. also some are overlooked and others grow poorly if deeply embedded in agar.

77

What is the purpose of performing the spread, streak, and pour plate technique?

to obtain a culture of isolated colonies

78

List the materials needed and describe how you would use them in performing the spread plate technique.

Broth stock culture, spreader, agar plate, loops
Take a loop of stock culture, transfer to the center of the agar plate, and spread it evenly over the surface with a spreader.

79

Lis the materials needed and describe how you would use them in performing the streak plate technique:

1. take a loop of stock culture and transfer to the edge of the 1st quadrant.
2. Strak out over the surface of the 1st quadrant with a zig-zag pattern.
3. Using same loop, streak out into the 2nd quadrant, with a zig-zag pattern.
4. Continue to zig-zag in the 2nd quadrant.
5. Continue in teh same way for the 3rd and 4th quadrants
at each quadrant flame the loop.

80

Why is it important to invert the petri plates during incubation?

This is to stop condensation from forming on the top of the lid
Condensation causes two problems (1) It will allow contamination to enter into the dish by moving accross the water film from the outside (2) It can create a tight seal and prevent oxygen entering the petri dish making it go anerobic.

81

What does CFU stand for? Define CFU?

Colony Forming Unit.Represents a pure colony forming from a single cell.
# of colonies grown during incubation

82

gram stain procedure

1. apply crystal violet (primary stain)
2. rise w/water (stains the cell purple)
3. apply mordant gram iodine (1 min) forms an insoluble bond with gram +
4. rinse w/water
5. decolorize w/ethyl alcohol (washes primary stain out of gram - ) so gram+ is purple, gram- is clear
6. counterstain w/safarin
7. rinse w/water
gram- will be pink, gram+ will remain purple

83

acid fast procedure

1. apply carbolfushin (primary stain)
2. rinse w/water (5 min0
3. decolorise w/acid alcohol (20 sec) non acid fast bacteria decolorises while acid fast remains pink
4. apply counterstain methylene blue (30 sec). provides contrast to entire acid fast and background material
acid fast are pink and non acid fast are blue

84

what are basic stains used for

used in staining because these dyes can bind to the slightly negative charge associated with bacterial cell wall

85

ex of gram +

staphyloccus, proteus vulgaris

86

ex of gram -

e coli

87

what is safrin used for

it detects the gram- cell walled bacteria

88

ex of acid fast bacteria

mycobacterium, mycobacterium tuberculosis, myobacterium leprae

89

acidic stains are

(-) charge, stains the entire background

90

basic stains are

(+)charge , stains the inside of the bacteria cell due to its neg charge inside

91

which acidic or basic stains the bacterial capsule

neither becox they are non ionic, neither stains will adhere to their surface

92

which procudure is crystal violet stain used for

in gram stain
it is a primary stain so it stains both gram+ and gram- purple

93

safrarin stains what color in gram+/-

gram+ purple
gram- pink

94

purpose of alcohol in gram stain

it dissolves the cell membrane and decolorises gram- bacteria due to peptidoglycan layer

95

shape of gram+
shape of gram-

gram+ is cocca (circle)
gram- is bacillus (rods)

96

acid fast and non acid fast stains are what color

acid fast stain retains the 1st stain (carbofushin) and is bright red
non acid fast stain retains 2nd stain (methylene blue) and is blue color

97

cell wall of gram+, affects of alcohol

thick peptidoglycan, that resists washing off by alchol

98

cell wall of gram-, affects of alcohol

thin peptidoglycan, easily destroyed by alcohol, so with any stains it gets washed off.

99

name some acidic stains

1 congo red
2 nigrosin
3 picric acid
4 safarin

100

name some basic stains

1 crystal violet
2 methylene blue
3 carbofushin
4 basic fushin
5 maliche green

101

types of differential stains

1 gram stain
2 capsule stain
3 spore stain
4 acid fast stain
5 flagella stain

102

which microorganism would have nuclei

all eurkaryotes such as fungi, algae, protozoa(largest nuclei)

103

what is the major disadvantage to stain bacteria

it kills bacteria so cant observe the behaviour of organism

104

what is the advantage of viewing live organism

so you can see how they move and interact in a more realistic enviornment

105

what is brownian movement

random collisions with water molecule. not a true motility

106

what type of movement would you expect to see if you stained the cells prior to doing the hanging drop

no movement becoz staining kills the bacteria

107

advantage of using depression slide for motility

allows bacteria to freely move around instead of getting stuck on normal flat slide

108

staining is mainly done for waht

contrast and morphology

109

which procedure was carbofushin used for

acid fast stains

110

why did we heat the slide when staining with carbofusin

heat allows the molecules of carbofusin to move faster and with energy that can penetrate the acid fast membrane

111

what part of bacterium is repsonsible for acid fast staining

mycolic acid that surrounds the bacterias membrane

112

what is the purpose of spore stain

to observe endorspores and spores produced by some species of bacteria

113

microorganism living in harsh conditions, that are dormant and reisistant are identified by the presence of what

endospores

114

what is the color of an endospore

green

115

why is it not necessary to heat the slide for safranin stain

so safarin doesnt go into the spores. heat is used to allow the first color to go into the spore and the second stain to color the surrounding cell

116

how does the ability to produce endospore affect the bacteriums abiltiy to cause disease

it allows them to last in harsh conditions until they can find another enviornment to reproduce and spread again

117

when are spores prodced

only when conditions are harsh

118

function of bacteria capsule

provide adherence
and
aids in avoiding phagocytosis or microphages

119

what is the purpose of capsule stain

to observe the slimy layer or capsule that surrounds bacteria.

120

stains used in capsule stain

klebsiella pneumnoniae
india ink
cyrstal violet

121

materials used in spore stain

plate culture of bacillus megaterium
electric hot plate and small beaker
spore stains, filtered malachite green and safarin

122

materials used for acid fast stain

mycobacterium,
staphylococcus aureus
hot plate
racks and beaker
carbolfushin, acid alchol, methylene blue

123

types of endospores (names)

bacillus megaterium - food posioning
bacillus cereus- food poisoning
bacillus anthracis - anthrax
bacillus sublitis - intestine
more deadly ones are
clostridum tatami - tetanus
clostridum botulinum -botulism and food poisoning
clostridum perfringens - gas, infected wounds

124

last layer of endospores

exosporium

125

first layer of endospore

plasma membrane

126

what stains the background in capsule stain

india ink stains the background purple

127

why do we stain only the background in capsule stain

becoz it the cells wont stain becoz of slimy capsule , but background can

128

what is contamination

anything that dont belong, presence of minor, unwanted materials

129

what are the sources of contamination

1. surface
2 air
3 lack of proper sterlization
4 incorrect sterile technique
5 cross contamination ( working wit 2 diff experiments taht got mixed)

130

agar is extracted from?

seaweed

131

pure form of agar is

agarose

132

melting point of agar

higher than 120 degree celcius

133

when does agar BEGIN to soldify

lower than 50-60 degree Celcius

134

when does agar become semisolid

lower than 25 degree celcius

135

What do nutrients provide for microbes

materials for production of energy and basic building blocks

136

what are heterotrophs

obtains nutrients from organic material such as glucose amino acid

137

what are autotrophs

self feeders, survive on inorganic molecules only or with light ex; K+, NA+, ca2+, fe2+

138

what is Browmian movement

random collision with water molecule. not a true mmotiltiy

139

what is the purpose of using alchol in the Gram stain?

alcohol dissolves the cell membrane and decolorizes gram negative bacteria due to its thin peptidoglycan layer

140

which bacterium is gram positive (color?)

purple stain, (cocca/round)

141

which bacterium is gram negative (color?)

red/pink (bacilli/rods)

142

what color is acid fast stain

red ex mycobacterium smegmatis

143

what color is non acid fast stain

blue ex: staphyloccus aureus

144

why do we heat the slide in acid fast stain when staining with carbofushin>

becuase the stain penetrates the outer layer and sticks to it

145

facts about endospores

1. highly resistant to most traditional methods of staining.
2. resistant to heat, dehydration, dessication, radiation, enzymatic and chemical attack

146

ex of endospores

bacillus and clostridium
clostridium are more deadly

147

what is the role of bacterial capsule in diseases

protects the cell from chemicals that could kill it. It helps them move from harsh condition

148

what stain stains the background in spore stains

india ink

149

what is the purpose of crystal violet stain in spore stain

to the stain the cell and be able to differentiate the slime from the cell

150

why are we not staining the capsule directly in spore stain

becoz it wont stick due to slime capsule

151

what is known ingredients reffered to as

synthethic or defined media
(formulated to exact specification)

152

what is unknown complicated mix reffered to as

complex or undefined nutrient media\\
(undefined ingredients are unknown in exact proportion)

153

general culture or isolation of unkown organisms is

complex nutrient media

154

specify isolation of particular types of organism is

selective media

155

diagnosis or identification of the types of microbe is

differentail media

156

ingredients needed for bacterial growth are

carbon, nitrogen, phosphorus, sulfur

157

carbon source come from

glycerol and alchols

158

nitrogen sources come from

amonium salt and nitrates

159

phosphorus needed for

required for amino acid and nucleic acid

160

sulfur found in

amino acid and nucleic acid

161

name a type of solid agar

tryptic soy agar

162

name a type of liquid agar

TSB broth

163

TSA Is (tryptic soy agar/solid form)

non fastidious microorganism and are non selective
shorter period, for counting and transferring

164

TSB is (broth liquid for agar)

can grow in huge volume and bigger batches

165

name the 2 types of culture

selective
differential

166

difference between selective and differential

selective - a medium that selects for the growth of a particular microorganism ex; high salt
differential - allows a more specific identification of certain bacteria ex; color changes or shade changes

167

MSA (mannitol salt agar) is it selective or differntail

is selctive and differentail

168

how is MSA selective and differentail

selective becoz it discourages the growth of most gram- organism and encourgages the growth of staphyloccus species
differentail becoz it contains the pH indicator phenol red

169

EMB (eosin methylene blue) is selective and differnetail why

selective becoz it isolates gm- coliform bacteria that are able to ferment lactose
differntial becoz it contains methylene blue which is toxic to most gm+ bacteria and contains bile salt which inhibits te growth of gm+ bacteria

170

what are the differnt types of medium for dilution theory

streak plate - quick and dirty
spread plate - more qunatitative
pour plate

171

calculaitons needed for dilution theory

DF - dilution factors
CFU- colony forming units

172

what is the purpose for streaking for isolation

to obtain isolated colonies

173

what is the purpose of flaming the loop when doing the streak plate for single colony isolation

it ensures that the loop starts clean and only the small amount of bacteria is useed to inoculate the next quadrant

174

why is pour plate NOT useful for isolation of pure culutres

becoz if the agar is not the right temperature results will not be accurate. some settle on bottom of agar

175

why are colonies of one type ususally bigger when they are not close to other ones

becoz they have more room to grow

176

phases of growth

1. lag pase
2. log phase
3. stationary phasee
4. decline or death phase

177

what is the lag phase

its the first phase
turning on enzymes to adjust to new enviornment (TIME)

178

what is log phase

its the 2nd phase
exponential growth with unlimited nutrients
(rapidly dividing)

179

what is the stationary phase

its the 3rd phase
limiting nutrients and waste accumlating, limited growth
(slows down)

180

what is the decline or death phase

its the 4 and last phase
Death surpass growth due to lack of nutrients and excess waste
(death/downfall)

181

what is growth requirements

1. light
2. hydrostatic pressure
3. temperature
4. pH
5. water
6. oxygen

182

bacteria that live in -15c to 10c

psychrophiles
extreme cold

183

bacteria that live in 20c to 30c

psychotrophs

184

bacteria that live in 25c to 45c

mesophiles

185

bacteria that live in 45c to 70c

thermophiles

186

bacteria that live in 70c to 122c

hyperthermophiles/archaea
extreme hot

187

how does temperature affect the bacteria

it affects the shape of enzymes, each design to fold to active shape at different temp. depending on whether they are thermophiles, mesophiles or psychrophiles

188

what does pH affect bacteria

it affects the integrity and molecular structures

189

how does concentration of water affect bacteria

osmolartiy and moisture conditions affect shapes as well as collision

190

how does oxygen affect bacteria

some bacteria require oxygen for growth

191

how do toxic oxygen radical form

from breakdown of oxygen molecules

192

areobic vs anaerobic

aerobic requires oxygen, anaerobic dont

193

obligage aerobes

require atmosphere levels of oxygen
(stay on top)

194

aerotolerant

dont use oxygen but can tolerate atmospheric levels of oxygen
(anywhere)

195

microaerophilic

need and tolerate less than atmospheric levels of oxygen
(stay in middle)

196

faculatative anaerobes

can live with or without oxygen
(all over, some on top, some on bottom)

197

obligate anaerobes

dont need oxygen and will be killed by the presence of oxygen
(stay on bottom to avoid oxygen)

198

toxic oxygen radicals

are harmful to bacteria becoz they cause damage. Toleration of the oxygen require special enzymes called superoxide dismutase. This converts oxygen radicals and water to hydrogen peroxide. Catalase converts hydrogen peroxide to harmless water and oxygen.

199

what is selective

selects/permits for growth of 1 type of microbes while inhibiting the growth of other

200

what is differential

permits us to distinguish or differentiate types of microbes which do grow

201

what is complex

it is a culture media where the ingredients are unknown in an exact proportion
exact compostion is unkown

202

what is defined

it is a culture media formulated to exact specifications
the exact composition is known.

203

what organisms grow on selective media

gram-

204

what organisms grow on differential media

ferment vs non ferment

205

why does s. aureus change color to yellow in msa plate

becoz it ferments manittol, producing an acid becoz of low ph and changes color to acidic color which is yellow

206

inhibiton of nonstaphlicocuus on msa plate will be on what condition

if sodium chloride is higher than 7.5%,
no growth due to high salt concentration, only s.aureus is capble of that becoz it ferments