Bacterial Visualization, Growth and Control Flashcards Preview

Microbiology > Bacterial Visualization, Growth and Control > Flashcards

Flashcards in Bacterial Visualization, Growth and Control Deck (112)
1

resolution is more important than _____

magnification

2

stain increases ____________

contrast

3

contrast increases _______

resolution

4

visualizing bacteria depends on (3)

magnification, contrast, resolution

5

sample fixation (def)

secures sample to slide before staining
kills sample
eliminates movement

6

sample fixation types

heat fixation
chemical fixation

7

heat fixation

dry sample on slide, pass sample/ slide through flame
denatured proteins in sample adhere sample to slide

8

chemical fixation

less damaging that heat
use for delicate samples (flagella, formalin)

9

basic stain

most common
positive ion colored
binds negatively charged cell surface

10

acidic (negative) stain

negative ion colored
stain repelled from negatively charged cell surface
ink darkens background around cell to show cell shape, arrangement

11

basic stain examples

safranin, carbolfuschin, crystal violet, methylene blue, malachite green

12

acidic (negative) stain example

nigrisin
india ink

13

mordant

not a stain
iodine (negative ion, heat

14

mordant function

increases affinity for stain, enhancing cell staining
increases visibility of external cell walls, flagella

15

staining techniques (3)

vital
simple
differential

16

vital stain

stains living sample
adds color to wet mount and hanging drop preparations
no heat fixation

17

simple stain

1 stain
stains all cells same color
methylene blue, crystal violet

18

differential stain

distinguishes 2 different species in sample
2 stains

19

differential stain types (5)

Gram stain
acid fast stain
endospore stain
capsule stain
flagella stain

20

4 steps to a differential stain

1. primary stain- stains all species same color
2. mordant
3. destaining
4. counterstain

21

Gram stain

distinguishes Gram positive bacteria (blue) from Gram negative (pink)

22

Gram stain steps

Start with heat fixed sample.
primary stain- crystal violet 1 min, rinse H20, mordant- iodine 1 min, rinse H20, Destain with acetone 5-10 seconds, rinse with water. Counterstain with safranin 3 min, rinse with water. Blot.

23

acid fast stain

stains Mycobacterium red
all others blue after counterstain

24

acid fast stain steps

primary stain carbolfucshin, heat slide to steaming (mordant) destain with alcohol- only Mycobacterium retain red. Counterstain with methylene blue.

25

endospore stain

stains endospores in their resistant stage, able to survive harsh conditions
ex Bacillus, Clostridium
endospores appear blue/green inside of pink cells

26

endospore stain steps

primary stain malachite green, heat (mordant) destain with water, counterstain with safranin

27

capsule stain

shows shape, arrangement of cells, thickness of capsule
ink is repelled from capsule surface so the cells appear white against black background
Klebsiella, Cryptococcus

28

capsule stain steps

primary stain India ink, counterstain methylene blue

29

flagella stain

requires gentle chemical fixation without heat
shows number and arrangement of flagella

30

pure culture

contains single species, not a mixture
derived from a single cell
colonies must not touch other colonies on plate to be certain culture is pure

31

3 techniques to obtain isolated colonies

streak plate
pour plate
spread plate

32

streak plate

easiest technique

33

pour plate

sample mixed in liquid agar
poured and allowed to harden

34

spread plate

sample poured on hardened agar
spread with sterile glass rod

35

culture media definition

solid or liquid containing nutrients or other agents required for growth

36

agar

polysaccharide from red algae

37

defined medium

known chemical composition

38

minimal defined medium

contains just enough nutrients to support growth

ex E. Coli mostly self-sufficient
Leuconostoc very demanding, requires many substances to grow

39

rich defined medium

excess nutrients
grows largest number of species

40

undefined (complex) medium

exact composition unknown, grows all species
contains: beef, blood, casein, yeast, soybeans

41

undefined medium examples

tryptic soy agar (TSA)
nutrient agar

42

selective medium

favors some species while inhibiting others
ex- salt agar isolates salt tolerant species from mixed sample
Staphylococcus

43

salt agar

isolates salt tolerant species from mixed sample
Staphylococcus

44

differential medium

growth characteristics on agar distinguish different species
ex- blood agar

45

blood agar

all species grow, colonies look different on blood

46

beta hemolysis

complete breakdown of red blood cells

47

alpha hemolysis

partial breakdown of red blood cells

48

gamma hemolysis

no effect on red blood cells, no hemolysins

49

mobility agar

all species grow
distinguished by movement in agar

50

selective and differential agar

selects some, differentiates by appearance

51

mannitol salt agar

selects for salt tolerant species
inhibits salt intolerant species
yellow color- Staphylococcus aureus

52

MacConkey agar

selects for Gram positives, inhibits Gram negatives
differentiates colonies by lactose fermentation- pinkish to reddish color

53

E. coli in MacConkey agar

ferments lactose
forms reddish colonies

54

Shigella and Salmonella in MacConkey agar

do not ferment lactose
form white colonies

55

enrichment procedure

expose mixed sample to unusual treatments

56

endospore isolation

boil sample, only spore formers survive

57

environmental conditions that directly influence bacterial growth

temperature, pH, oxygen

58

temperature maintenance

incubators, water baths, refrigerators

59

psychrophiles

cold tolerant bacteria

60

mesophiles

room temperature loving bacteria

61

thermophiles

heat tolerant bacteria

62

E. coli optimum growth

37 C
proteins begin to denature at 40 C+

63

pH

measure of hydrogen ions in solution

64

fungi pH

4.5- 6.0

65

blood pH

7.4 (7.2 and 7.6 are toxic)

66

buffers

prevent abrupt changes in pH
donate or remove Hydrogen ions in solutions
ex phosphate salts, calcium carbonate added to culture

67

oxygen manipulation techniques (3)

thioglycolate
Brewer anaerobic jar
candle jar

68

thioglycolate

establishes oxygen gradient in culture
bottom of tube anaerobic

69

Brewer anaerobic jar

combines oxygen with hydrogen to form water
anaerobic

70

candle jar

burning candle reduces oxygen concentration
microaerophilic

71

strictly aerobic

require atmospheric levels of oxygen
Pseudomonas aeriginosa

72

obligate anaerobic

do not use oxygen
killed by oxygen
Clostridium

73

faculative anaerobic

use oxygen if present but can live without it
E. coli
Staphylococcus aureus

74

aerotolerant anaerobic

do not use oxygen but not harmed by it
Lactobacillus

75

microaerophiles

require oxygen but less than atmospheric
Neisseria sicca
Micrococcus

76

Bacterial growth limiting factors

physical- temperature, pH, osmotic pressure
chemical- all molecules required for growth and reproduction
C, O, N, S, P, trace elements, organic growth factors

77

bacterial growth curve phases

lag, log (exponential), stationary, death (decline)

78

lag phase

onset of colony formation from 1 cell or few cells
length depends on inoculation source, amount of resources available

79

log (exponential) phase

begins with few cells, optimal conditions and abundant resources
exponential growth characterized by rapid doubling time

80

log phase limitations

environment changes as numbers increase
nutrients are depleted, pH changes (more acidic), toxins increase
growth rate declines, levels off

81

stationary phase

population size remains constant
number of cells added equals number of cells dying
cell metabolism shifts from reproduction to survival
lasts indefinitely if minimum resource levels can be maintained

82

carrying capacity

maximum number of individuals environment will support

83

death (decline) phase

resources eventually become limited
existing cells die off at a faster rate than new cells are added

84

targets of control treatments

external cell wall, cell membrane, proteins, nucleic acids (DNA, RNA), ribosomes, enzymes

85

bacterial control methods

disinfection/ sterilization, physical/ chemical

86

disinfection

microbiostatic- does not completely eliminate bacteria
inhibits or prevents growth- low numbers not disease causing
decontamination, antisepsis

87

decontamination

remove bacteria from surfaces

88

antisepsis

disinfection of living tissue

89

sterilization

microbiocidal; kills all microbes including endospores

90

physical controls

heat
refrigeration
radiation
osmotic treatment
filtration

91

heat treatments

moist heat
dry heat
flame sterilization
pasteurization

92

moist heat

steam
autoclave

93

dry heat

oven
less effective than moist heat

94

flame sterilization

aseptic transfer of organisms

95

pasteurization

briefly expose perishable fluids to high heat

96

flash pasteurization

continuous, high temp, short time
milk 72C for 15 seconds- kills all pathogens

97

continuous ultra high temp pasteurization

140 C for 1 to 3 seconds
may affect taste

98

batch (vat) pasteurization

63C, 30 min

99

superheated steam

sterilization
store liquids at room temperature without spoiling
product may degrade- dairy coffee creamer

100

refrigeration

low temp does not kill but slows metabolism, reproduction

101

radiation

damages DNA and denatures proteins

102

radiation types

ionizing
non ionizing
microwaves

103

ionizing radiation

high energy, penetrating, X rays, gamma rays
penetrates covers- food, medical supplies, mail

104

non ionizing radiation

lower energy, non penetrating, uv light
does not penetrate covers- wrappers, surfaces
requires direct exposure to radiation

105

microwaves

less effective than bacteria
longer wavelength, lower energy

106

osmotic treatment

high solute concentration (salt, sugar)
create hypertonic environment to remove water from cells but do not kill cells

107

filtration

filters remove most bacteria- does not remove viruses
sterilize liquids or gases damaged by heat

108

chemical treatments

kills bacteria or reduces numbers to low levels

109

chemical treatment examples

bleach- kill bacteria on slides; pipettes
lysol- clean bench
isopropanol- kills bacteria, cleans surface, evaporates quickly
soap- emulsifies lipids, helps remove bacteria from surfaces
ethylene oxide (gas)- deeply penetrating, requires more time; used to sterilize space craft returning from moon and Mars, sterilize medical equipment

110

effectiveness of control treatments depends on

-characteristics of organism being treated
-number of cells in colony and growth stage colony is in
-organic substances in environment may interfere with treatments
-temperature and length of exposure time

111

D-value

decimal reduction time
measures rate of decline in response to heat treatment
time in minutes required to kill 90% of population at given temperature
90% die in 10 minutes- 90% of remaining 10% die in next 10 minutes

112

Microbial death graph

higher temp, shorter time