Bacterial Visualization, Growth and Control Flashcards
(112 cards)
1
Q
resolution is more important than _____
A
magnification
2
Q
stain increases ____________
A
contrast
3
Q
contrast increases _______
A
resolution
4
Q
visualizing bacteria depends on (3)
A
magnification, contrast, resolution
5
Q
sample fixation (def)
A
secures sample to slide before staining
kills sample
eliminates movement
6
Q
sample fixation types
A
heat fixation
chemical fixation
7
Q
heat fixation
A
dry sample on slide, pass sample/ slide through flame
denatured proteins in sample adhere sample to slide
8
Q
chemical fixation
A
less damaging that heat
use for delicate samples (flagella, formalin)
9
Q
basic stain
A
most common
positive ion colored
binds negatively charged cell surface
10
Q
acidic (negative) stain
A
negative ion colored
stain repelled from negatively charged cell surface
ink darkens background around cell to show cell shape, arrangement
11
Q
basic stain examples
A
safranin, carbolfuschin, crystal violet, methylene blue, malachite green
12
Q
acidic (negative) stain example
A
nigrisin
india ink
13
Q
mordant
A
not a stain
iodine (negative ion, heat
14
Q
mordant function
A
increases affinity for stain, enhancing cell staining
increases visibility of external cell walls, flagella
15
Q
staining techniques (3)
A
vital
simple
differential
16
Q
vital stain
A
stains living sample
adds color to wet mount and hanging drop preparations
no heat fixation
17
Q
simple stain
A
1 stain
stains all cells same color
methylene blue, crystal violet
18
Q
differential stain
A
distinguishes 2 different species in sample
2 stains
19
Q
differential stain types (5)
A
Gram stain acid fast stain endospore stain capsule stain flagella stain
20
Q
4 steps to a differential stain
A
- primary stain- stains all species same color
- mordant
- destaining
- counterstain
21
Q
Gram stain
A
distinguishes Gram positive bacteria (blue) from Gram negative (pink)
22
Q
Gram stain steps
A
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
Q
acid fast stain
A
stains Mycobacterium red
all others blue after counterstain
24
Q
acid fast stain steps
A
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
