Microbiology Labs Week 3-5

Front 1

Streak Plate

Back 1

Isolation technique of a mixed sample

Front 2

How to do a streak plate?

Back 2

1. streak over a plated agar medium
2. 4 streaks (flame the loop after each streak)

Front 3

Purpose of Streak plate

Back 3

during streaking, the cell density decreases
colonies --> individuals (isolation)

Front 4

Streak plate success?

Back 4

Cells that have been sufficiently isolated will grow into colonies of ONLY the original cell type

Front 5

Colony-forming unit

Back 5

CFU

Front 6

CFU?

Back 6

colonies form from individual cells & others from pairs, chains or clusters

Front 7

Streak plate method form what?

Back 7

individual colonies on plates

Front 8

Why use Streak plate method?

Back 8

CFU transferred to a sterile medium to start a pure culture

Front 9

Zigzag inoculation of Agar plates with cotton swab when?

Back 9

have pure cultures.
when sample does not have high cell density

Front 10

Innoculation of agar plates with cotton swab in prep for Quadrant Streak plate:

Back 10

1. preformed as initial streak for isolation (streak 1).
2. done with mixed cell culture with high cell density
3. streak Q1 with swab; use loop Q2-4

Front 11

What is Steam sterilization?

Back 11

Autoclave

Front 12

Why is Steam Sterilization used?

Back 12

most effective and most common way to sterilize contaminated items

Front 13

Increase the atmospheric pressure and what happens to the boiling temperature?

Back 13

It increased, but the cook time shortens

Front 14

Autoclave specifics

Back 14

121*C
15 psi (pressure/square inch)
15 min
super heated steam under pressure to kill heat-resistant organisms

Front 15

SDA

Back 15

sabourand dextrose agar
good media for fungi
acidic
pH 5.6
lower pH = lower bacteria

Front 16

TSA

Back 16

tryptic soy agar
enriched media
pH 7
good for bacteria growth

Front 17

Heat resistant organisms

Back 17

spore producing (in protective form) Bacillus, Geobacillus & clostridium

Front 18

Sterilizing Temp.

Back 18

121-127*C (250-260*F)

Front 19

Sterilizing time

Back 19

Vary to size & consistency of material
Items must reach optimum T*C for 15 min.

Front 20

What is used as good indicators that sterilization is complete?

Back 20

special thermometers
colored-coded autoclave tape

Front 21

What is a Biological indicators?

Back 21

determine sterilization
device containing a known pop. of viable organisms (significant & quantified resistance to sterilization) used to validate autoclave

Front 22

What is used as a typical biological indicators

Back 22

bacterial spores
Dormant form of an organism, highly resistant to chemical & physical changes
If not killed, they will germinate & ferment => lowers pH (acidic) purple --> yellow

Front 23

Standard Plate Count

Back 23

Viable Count
Indirect Count
Live cells ONLY
Estimate of actual living cells in a sample

Front 24

Standard Plate Count allows?

Back 24

An estimate of pop. density in a liquid sample by plating dilute portions & counting # of colonies formed (CFU)

Front 25

CFU

Back 25

Number of Colonies Formed
# of cells

Front 26

Serial dilution

Back 26

begin with unknown concentration (density) of cells-> dilute with a few cells

Front 27

Countable plates

Back 27

30 - 300 colonies (CFU)
Above 300 (too many too count; too many to be viewed as individual colonies)
Below 30 (too few to count; statistically unreliable)

Front 28

In dilutions

Back 28

V1D2 = V2D2

D2 = (V1xD1) / V2
= (1.0 mL x 10^-1) / 10 mL

Front 29

Plate Spread

Back 29

Known volume of dilution
Count colonies

Front 30

OCD

Back 30

original cell density

OCD=CFU/Dil'n x Vol.transfered to plate

Front 31

OCD express in what units?

Back 31

CFU/mL

Front 32

Volume of original sample

Back 32

is the product of vol. transferred & the dilution of the tube it came from.

Front 33

Direct Counts

Back 33

take a small portion of a sample & use the data gathered from it to calc. overall pop. cell density

Front 34

Petroff-Hausser counting chamber

Back 34

-microscopic slide with 0.02mm deep chamber ("well") in center with grid
-25 large squares (16 small squares in each) = 400
-1 square mm
well (when covered with cover glass & filled with suspension cells) vol. =5 x 10^-8 mL
-each small square = 50,000 avg. size cocci cells

Front 35

OCD

Back 35

original cell density = cell density

Front 36

OCD used for?

Back 36

counting cells found in a predetermined group of small squares & dividing by (# of squares)(Dil'n)(Vol)

Front 37

OCD calculation

Back 37

(Cells counted)
__________________________
(# of Squares) (Dil'n) (Vol.)

Front 38

Optimum density

Back 38

5-15 cells/small square
*For accuracy recommend min. overall count of 600 cells

Front 39

Indirect Count Advantages

Back 39

1.Works well for cells that separate in a short amt of time after they divide

Front 40

Indirect Count Disadvantage

Back 40

1. Doesn't work well for cells that stick together after cell division
2. Mixed samples have diff. nutritional needs
3. Sample error = uneven distribution of the sample on the agar

Front 41

Direct Count Advantages

Back 41

Faster

Front 42

Direct Count Disadvantages

Back 42

There must be a certain # of organisms b/f there are enough to be seen (both viable & nonviable count)

Front 43

Spread plate technique

Back 43

method of isolation in which a diluted microbial sample is deposited on an agar plate & spread uniformly across the surface with a glass rod

Front 44

How to tell if a sample it properly diluted?

Back 44

With a properly diluted sample, cells (CFUs) will be deposited far enough apart on the agar to grow individual colonies

Front 45

Consequences of not spreading the inoculum adequately?

Back 45

*uneven distribution, so
what try to grow/inhibit = no isolated colonies

Front 46

Separate a mixed culture

Back 46

separate, isolate, & harvest out = streak method

Front 47

Optical Density

Back 47

OD
*Used to measure the bacteria concentration in a suspension
by measuring the amt of light scatter

Front 48

What is the correlation b/w the OD & cell # for your culture?

Back 48

*Greater the scatter = more bacteria present
*Greater the dilution = the smaller amt of light scatter occurs

Front 49

Why do you plate count (viable) & OD measure?

Back 49

data will give us exact measurements of bacteria concentration in our culture for graphing

Front 50

Determining the Growth by Optical Density uses what? quantify what?

Back 50

Spectrophotometer
Quantify the microbial population in a sample

Front 51

Bacteriophages

Back 51

Phages
Viruses that attack bacteria

Front 52

How do phages kill bacteria?

Back 52

1. Some viruses attach to the bacterial cell wall
2. inject viral DNA into the cytoplasm
3. Viral genome commands the cell to produce more viral DNA & viral proteins, used to assemble more phages
4. Phage assembly completed, the cell lyses & releases the phages
5. Phages attack other bac. cells

Front 53

Lytic cycle

Back 53

1. Some viruses attach to the bacterial cell wall
2. inject viral DNA into the cytoplasm
3. Viral genome commands the cell to produce more viral DNA & viral proteins, used to assemble more phages
4. Phage assembly completed, the cell lyses & releases the phages
5. Phages attack other bac. cells

Front 54

Lysis of bacterial cells on agar plate produce what?

Back 54

A clearing
Plaques

Front 55

Plaques

Back 55

Clearing
Areas where virus has killed bacteria

Front 56

Plaque assay used for what?

Back 56

Means of calculating the phage concentration in a given sample

Front 57

Plaque assay

Back 57

1. Sample of bacteriophage (diluted by serial dil'n) added to a plate inoculated with bacterial host to produce a lawn of growth
2. # of plaques formed used to calculate the original phage titer (density)

Front 58

Titer

Back 58

Density

Front 59

Phage titer

Back 59

phage density

Front 60

Plaque assay technique

Back 60

1. serial dil'n to produce countable plates

Front 61

Plaque assay uses what technique

Back 61

Pour-plate technique

Front 62

Pour-plate technique

Back 62

bacterial cells and viruses are first added to molten agar and then poured into the plate

Front 63

What does the pour-plate technique do for the plaque assay?

Back 63

*diluted phages added to exposure time to bacteria = preadsporption period
*Then this phage-host mix is added to a tube of soft agar, mixed, & poured onto prep Nutrient Agar plates as an agar overlay

Front 64

Preadsporption period allows for what?

Back 64

allows the virus to attach to the bacterial cells

Front 65

Agar overlay

Back 65

This technique allows you to produce a homogeneous lawn of bacteria within a thin layer of agar across the surface of a plate. Bacteria are added to a soft top agar (0.75% agar, as opposed to the usual 1.5% for agar plates) which has been melted at 100°C and cooled to 45°C. This is warm enough so the agar remains liquid, but cool enough so that the bacteria are not killed (for a period of time). The melted agar/bacterial suspension is mixed and poured evenly across the top of an agar plate and allowed to solidify.

Front 66

Why is agar overlay used in plaque assay?

Back 66

To produce a homogenous mixture of phages and bacteria;
Allow bacteria exposure to phages to occur
*when solidifies, the agar immobliizes the bacteria while allowing the smaller phages to diffuse short distances & infect surrounding cells

Front 67

Plaques appearing on a plate is an indication of what?

Back 67

Bacteria infected with viruses;
the bacteria cells have been lysed by the virus

Front 68

Countable plates

Back 68

30 -300 plaques
Same as standard plate count

Front 69

PFU

Back 69

plaque forming units

Front 70

Phage titer

Back 70

original phage density

Front 71

Phage titer calculation

Back 71

Phage titer = (PFU) / (Volume plated x Dilution)
*PFU / Original sample volume
PFU/mL

Front 72

What bacteriophage was used to kill Escherichia coli?

Back 72

T4 coli-phage

Front 73

T4 phage lytic cycle

Back 73

1. Attachment
2. Penetration
3. Uncoating
4. Synthesis
5. Assembly
6. Generalized Transduction
7. Release
Cycle can take 25 minutes
Can release a few hundred phage progeny

Front 74

T4 phage lytic cycle

Attachment

Back 74

Phage attaches by its legs to specific receptors on E. coli

Front 75

T4 phage lytic cycle

Penetration

Back 75

T4 process of injecting its DNA into E.coli
*Virus condenses
Entire phage genome is in the host

Front 76

T4 phage lytic cycle

Uncoating

Back 76

Removal of virus genome from its capsid
Phage DNA is being replicated

Front 77

T4 phage lytic cycle

Synthesis

Back 77

Phage DNA replicated in bacteria host
Synthesis of Virus proteins (to form virus parts)
Degrades host DNA (sometimes host DNA incorporated into phage DNA)

Front 78

T4 phage lytic cycle

Assembly

Back 78

Synthesis leads to assembly of the phage progeny, where capsid subunits come together to form the capsid into which the genome is inserted
& tail comes together & attaches to the capsid

Front 79

T4 phage lytic cycle

Generalized Transduction

Back 79

Host DNA is incorporated into new T4 phage DNA, giving it new genes
Genetic diversity increased

Front 80

T4 phage lytic cycle

Release

Back 80

Fully assembled phages are released as the cell bursts

Front 81

Why must there be enough bacteria inoculated to produce a lawn of growth?

Back 81

Plaque assays ID the presence of viruses by the absence of growing bacteria.
If not enough bac. on plate then there will be regions of no growth = false phage assay

Front 82

How might results be altered if skip preadsorption phase?

Back 82

False absence of phage, due to low plaque count.
Preadsorption allows time for the phages to attach to the bacterial cells (not time to kill them, just start the process)

Front 83

Why is the water bath set at 50*C?

Back 83

50*C = 122* F (shower, hot food)
Over 130*F (tissue damage)
Lower Temp. might be to cold for growth or slow growth (chemical rxns slower)
High temp. will denature proteins & kills speciment

Front 84

Zone of inhibition

Back 84

Phage kill zone of bacteria

Front 85

Cardinal Temperatures

Back 85

Each species is characterized by a minimum, maximum, and optimum temperature of growth
*Below min. the organism will not survive

Front 86

Optimum Temperature

Back 86

Temperature an organism shows the greatest growth over time
Highest growth rate

Front 87

Psychrophiles

Back 87

Grow only below 20*C
Common in ocean, Arctic, Antarctic habitats (permeant cold)
No fluctuation

Front 88

Psychrotrophs

Back 88

Adapted to cold habitats that fluctuate
0* - 30*C

Front 89

Mesophiles

Back 89

15* - 45*C

Front 90

Thermophiles

Back 90

Above 40*C
Composting organic material & in hot springs

Front 91

Obligate Thermophiles

Back 91

Will not grow below 40*C

Front 92

Facultative thermophiles

Back 92

Grow below 40*C

Front 93

Extreme thermophiles

Back 93

65* - 110*C
Bacteria isolated from hot ocean floor ridges
Grow best above 80*C

Front 94

Optimal Temperature of
Serratia marcescens

Back 94

37*C
(Mesophiles range 25-45)
At optimal Temp. produces a red pigment

Front 95

At what temperature will Serratia marcescens stop producing a red pigment?

Back 95

42*C (off-white)
The enzyme is beginning to b/m denatured and no longer works

Front 96

Why do different temperatures produce different growth rates?

Back 96

Enzyme activities are temp. dependent.
Higher temp. means higher activity (dentures/unravels the protein)
Lower temp. means lower activity (stops synthesis)

Front 97

pH

Back 97

concentration of hydrogen ions in a solution
*"pondus hydrogenii" = hydrogen power or hydrogen potential

Front 98

Soren Peter Lauritz Sorensen

Back 98

Danish biochemist
1909
invented pH range (0-14) based on logarithmic scale

Front 99

pH calculation

Back 99

pH = -log [H+]

Front 100

Pure water pH

Back 100

7
10^-7 moles of hydrogen ions per liter

Front 101

Acidophiles

Back 101

organisms adapted to grow well in environments below pH 5.5

Front 102

Neutrophiles

Back 102

pH 5.5-8.5

Front 103

Alkaliphiles

Back 103

pH 8.5

Front 104

Bacteria under normal circumstances

Back 104

maintain a near-neutral internal environment regardless of their habitat

Front 105

pH outside the normal range of the organism, what happens?

Back 105

destroy necessary membrane potential (in the production of ATP) and damage vital enzymes beyond repair

Front 106

Denaturing proteins/enzymes

Back 106

*conformational changes in the proteins' tertiary structure
Lethal to the ell

Front 107

Acids form from what fermentation?

Back 107

carbohydrate fermentation

Front 108

Alkaline products from from what?

Back 108

protein metabolism

Front 109

Hydrogen phosphate

Back 109

buffer
In solution can alternate b/w WA (H2PO4-) & conjugate base (HPO4-2)
Maintains H+/OH- equilibrium

Front 110

Why should you not connect data points on a graph?

Back 110

Indicates a continuous and that there is data inbetween the points are valid; they may not be
*only connect data points if the data is continuous, but it is changing all the time

Front 111

Water is the principal component of what part of the cell? Essential for what

Back 111

cellular cytoplasm
* Essential source of E+ & H+ions

Front 112

Turgor pressure

Back 112

Internal cellular pressure
Important in prokaryotes, like plants

Front 113

Plasmolysis

Back 113

Separation from the cell wall
*Prokaryotes require water to prevent shrinking of cell membrane resulting in separation from the cell wall

Front 114

Bacteria control their turgor pressure how?

Back 114

transporting in & maintaining high K+ & Na+ ion concentration
creating a [ ] gradient that promotes diffusion of water into cell

Front 115

Osmosis

Back 115

movement of water from an are of low solute concentration to an area of higher solute concentration

Front 116

Osmotic pressure

Back 116

the pressure which needs to be applied to a solution to prevent the inward flow of water across a semipermeable membrane.[1] It is also defined as the minimum pressure needed to nullify osmosis.

Front 117

Osmotic pressure refers to what?

Back 117

The ability of a solution to pull water toward itself through a semipermeable membrane

Front 118

Hyposmotic

Back 118

solution having low osmotic pressure
low solute concentration

Front 119

hyperosmotic solution

Back 119

solution having high osmotic pressure
high solute concentration

Front 120

Isosmotic

Back 120

a solution having osmotic pressure equal to that of the cell
Water will tend to move in both directions

Front 121

Halophiles

Back 121

grow optimally in [NaCl] of 3% or higher

Front 122

Extreme halophiles

Back 122

specialized cell membranes and enzymes that require salt concentrations from
15% - 25%
Will not survive in lower salinity

Front 123

Osmotolerant

Back 123

grow over wide range of salinities
Most bacteria live in [NaCl] less than 3%

Front 124

Germicides

Back 124

Substances/systems used to prevent the spread of pathogens
*specific in nature & typically include the name of the target pathogen ("tuberculocide," "virucide," "sporocide")
Most germicides are broad-spectrum
2 categories (decontamination, disinfection, sterilization)

Front 125

Decontamination

Back 125

germicides lowest level of control
"reduction of pathogenic microorganisms to a level at which items are safe to handle without protective attire"
*physical agents used, removal, cleaning
*sterlize must come in direct contact with pathogens

Front 126

Disinfection

Back 126

Next level of control
3 sublevels (low, medium, high)
Based on effectiveness against specific control pathogens (kill targeted pathogens, not spores)
*chemical sterilants (kill all vegetative cells & spores)
*Methods: dry heat, moist heat, & UV
*reduce/eliminate pathogens on/in living tissue = antiseptics

Front 127

Sterilization

Back 127

*Complete elimination of viable organisms, including spores
*highest level of pathogen control
*chemicals, gases, incineration, dry heat, moist heat, ethylene oxide gas, radiation, low-temperature plasm

Front 128

Electromagnetic energy

Back 128

X-rays, UV, Visible light, Infrared
Wavelengths of light

Front 129

UV light

Back 129

UV-A longest wavelength 315-400 nm
UV-B 280-315 nm
UV-C 100-280 nm

Front 130

Wavelengths

Back 130

the distance b/w adjacent wave crests and is typically measured in nanometers

Front 131

Which UV is detrimental to bacteria? why?

Back 131

UV-C (100-280nm)
Results in irreparable DNA damage & death of the organism
*Mutagenic effects of UV & DNA repair
Shortest wavelength = highest energy

Front 132

Why do endospores survive UV exposure?

Back 132

Spore-forming bacteria such as Bacillus are able to survive much higher doses of UV and other types of radiation. Spores can survive very harsh conditions (high/low temperature, low water, radiation, antibiotics, osmotic pressure, etc.) for extended periods of time before becoming active again when conditions improve.

Front 133

Endospores

Back 133

dormant, tough, and non-reproductive structure
It is a stripped-down, dormant form to which the bacterium can reduce itself. Endospore formation is usually triggered by a lack of nutrients, and usually occurs in Gram-positive bacteria. In endospore formation, the bacterium divides within its cell wall.
Endospores enable bacteria to lie dormant for extended periods, even centuries. Revival of spores millions of years old has been claimed.[2] When the environment becomes more favorable, the endospore can reactivate itself to the vegetative state
resistant to ultraviolet radiation, desiccation, high temperature, extreme freezing and chemical disinfectants.
The core contains the spore chromosomal DNA which is encased in chromatin-like proteins known as SASPs (small acid-soluble spore proteins), that protect the spore DNA from UV radiation and heat.

Front 134

Antibiotics

Back 134

natural antimicrobal agents produced by microorganisms
Penicillium notatum
*treat bacteria infections

Front 135

Antimcrobials/antimicrobics

Back 135

all substances used to treat bacterial infections that are synthetic

Front 136

Kirby-Bauer Test

Back 136

disk diffusion test
*valuable standart tool for measuring the effectiveness of antimicrobics against pathogenic microorganims
*Ab impregnated paper disks placed on plate inoculated to form bacterial lawn, incubated (to allow bac growth & time for Ab to diffuse into agar).
*as the drug moves through the agar, it established a [ ] gradient
*if an organism is susceptible to it = clear zone (around disk) of inhibited growth

Front 137

Zone of inhibition

Back 137

clear zone
If an organism is susceptible to it, growth will be inhibited
*depends on the sensitivity of the bacteria to specific Ab agent & MIC

Front 138

Minimum inhibitory concentration (MIC)

Back 138

*important to confirm resistance
the min. amt. of Ab to get results

Front 139

Bactericidal

Back 139

kill organism

Front 140

bacteriostatic

Back 140

stop growth

Front 141

Muller-Hinton agar

Back 141

pH 7.2-7.4
4mm depth (important b/c its effect on diffusion)
low levels of thymine & thymidine & controlled levels of Ca & Mg = required to produce a correct zone diameters with the Abs used
Store @ 2-8*C

Front 142

Factors that influence disk diffusion susceptibility tests

Back 142

Medium
Excess surface moisture on medium
Agar depth
Disc potency
Inoculum concentration
pH
B-lactamase production by test organisms

Front 143

Do not use plates if they show:

Back 143

Microbe contamination
Discoloration
Drying
Cracking
Deterioration
Excess shrinkage (desiccation) may lead to false suseptibility results

Front 144

How does depth affect diffusion of Ab?

Back 144

Thick agar = slows lateral diffusion & produces smaller zones than plates held to the 4mm standard

Front 145

Inoculation for Kirby-Bauer test is made with a broth culture diluted to match what?

Back 145

A 0.5 McFarland turbidity standard

Front 146

McFarland Turbidity Standard

Back 146

comparison of McFarland turbidity
Has a specific concentration of precipitated barium sulfate to produce turbidity
Reached b/f inoculating the plate

Front 147

McFarland standard

Back 147

0.5 McFarland
1.5 x 10^8 cells/mL

Front 148

Disk dispenser

Back 148

contains a specified amt of Ab agent (printed on disk) dispensed into plate

Front 149

Kirby-Bauer test Advantages

Back 149

Allows determination of the most effective Ab to disease

Front 150

Kirby-Bauer test Disadvantages

Back 150

takes 24 hr turn around
doesn't indicated the dose needed to kill/inhibit the pathogen long enough for the immune system to work

Front 151

Resistance mechanisms

Back 151

Break down in 3 main categories:
1. altered target such that the Ab no longer can interact with the cellular process
2. alteration in how the drug is taken into the cell
3. enzymatic destruction of the drug

Front 152

Antibiotics affect on cells

Back 152

1. attack cell wall
3. interfere with biosynthesis rxns

Front 153

Chloramphenicol
Cellular target

Back 153

prevents peptide bond formation during translation

Front 154

Chloramphenicol
Resistance mechanism

Back 154

1. poor uptake of drug
2. inactivation of drug

Front 155

Chloramphenicol

Back 155

bacteriostatic

Front 156

Ciprofloxacin
Cellular target

Back 156

interferes with DNA replication

Front 157

Ciprofloxacin
Resistance Mechanism

Back 157

1. Altered target
2. Poor uptake of drug

Front 158

Ciprofloxacin

Back 158

bacteriocidal

Front 159

Trimethoprim
Cellular Target

Back 159

inhibits purine and pyrimidine synthesis

Front 160

Trimethoprim
Resistance mechanism

Back 160

1. altered target

Front 161

Trimethoprim

Back 161

Bacteriostatic
S >16
I 11-15
R <10

Front 162

Penicillin
Cellular target

Back 162

inhibits cross-linking of the cell wall's peptidoglycan (b-lactam Ab)
Narrow spectrum

Front 163

Penicillin
Resistance mechanism

Back 163

1.altered target
2.poor uptake of drug
3.production of B-lactamases

Front 164

Penicillin

Back 164

Bacteriocidal
S >29
R < 28

Front 165

Chemical germicides

Back 165

reduce # of pathogens on a surface, in a liquid, or on/in living tissue

Front 166

Germicides

Back 166

designed for use on surfaces

Front 167

Disinfectants

Back 167

liquids
germicides designed for use on surfaces

Front 168

Antiseptics

Back 168

germicides designed for use on/in living tissue

Front 169

Use-Dilution Test

Back 169

standard procedure used to measure the effectiveness of disinfectants specifically against Staphylococcus aureus, Salmonella enteric serovar Cholerasuis, & Pseidomonas aeruginosa
*Standard procedure: glass beads exposed to bacteria, then germicides and medium to grow (test effectiveness of germicide)

Front 170

Sufficient germicide

Back 170

prevents 95% microbial growth
*meets required standard
*considered usable dilution of that germicide for a specific application if 95%