Lab Test 2

Question 1

Why is the isolation of organisms necessary?

Answer 1

• In order to characterize the growth or biochemical activities of bacteria, each organism must be separate from its companions.
• Without the isolation of the organism, two or more organisms could contribute to a growth pattern or reaction, but there would not be a distinction of which of the organisms was responsible*

*this was Pasteur’s problem when trying to prove the germ theory of disease

Question 2

Pure culture

Answer 2

A single species (or strain) of bacteria growing in a culture; essentially a colony of cloned cells which are all identical

Question 3

Isolation streak technique

Answer 3

To isolate an organism from other organisms in the culture.

Involves successively diluting an original inoculum of mixed culture over four sections drawn on an agar petri dish. This procedure aims to eventually spread the individual cells apart enough for separate colonies to grow from them by cloning.

If done correctly each quadrant of the dish will show progressively fewer colonies, until finally separate colonies are seen. (pure cultures)

Question 4

What are 2 ways that the number of bacteria on your loop are reduced during the isolation streak technique?

Answer 4

• Flaming

- Rubbing the loop on the agar

Question 5

Subculturing

Answer 5

The process of aseptically transferring a culture from one culture medium to another

Question 6

What is the purpose of subculturing?

Answer 6

• Maintenance: bacteria grow so rapidly that they deplete nutrients in their environment, as well as create toxic wastes which build up. Just as a pet cat needs fresh food, water, and cat liter bacteria in cultures need the same every so often so that they can remain alive for study. It is essential to keep a good stock culture of organism for testing
• In order to study the growth or biochemical characteristics, cultures must be placed in specific media or growth conditions to see results. Ex. to determine whether a streptococcus species can produce hemolysin it must be inoculated on a blood agar plate.

Question 7

Important things to remember during the subculturing technique:

Answer 7

-The tube containing the culture in the space between the ring finger and middle finger. The fresh new tube to be inoculated goes in the space between the middle finger and the pointer finger, parallel to the old tube.

Question 8

Five characteristics observed from a slant culture:

Answer 8

-Amount: large (grows to cover entire surface of slant), small (barely visible at the streak, having spread little, if any), moderate (in between two extremes)

• Color: Most is white, cream, or grayish. For some, the bacteria release a pigment which colors the agar
• Shape: May be straight, scalloped, tear dropped, or show a branching, tree like pattern.
• Opacity: Transparent (see through) , translucent (can see color through, blurry), opaque (cannot be seen through)
• Odor: e.coli - smells like poop, bacillus species tend to smell like fresh turned earth, pseudomonas has a distinctly grape like smell

Question 9

3 characteristics observed from a broth culture

Answer 9

-Do not support colony growth as seen on plates and slants
Turbidity: Uniform dispersion of the bacteria within the liquid will lead to turbidity, or cloudiness

Sediment: Some organisms will settle out or fall to the bottom of the tube. This will be seen by holding up the tube and looking at it from beneath.

Pellicle: Skim on the surface of the broth. Often seen with spore forming species, in which the pellicle only consists of spores. Mycobacterium also forms a pellicle, it is very waxy to begin with and does not emulsify well in water.

Question 10

5 characteristics of isolated colonies

Answer 10

• Size- Individual colonies vary in size
• Color - Growth may be a dull beige, gray or have vibrant colors
• Margin - May be smooth, jagged, scalloped, branching etc
• Shape - Most are round, but some are irregular, such as mycobacterium, or even oval
• Elevation - Flat or raised

MESS-C

Question 11

Characteristics of mycobacterium

Answer 11

Very waxy! will form large pellicle, irregular shaped colony

Question 12

What factors could affect the normal pattern and cause uncharacteristic growth patterns in a species

Answer 12

• If colonies are close together they may appear smaller due to crowding/competing for nutrients
• Temperature will cause the colonies to grow to different sizes
• Moisture - a lot tends to allow the colonies to grow larger
• Nutrient/waste levels
• Length of incubation

Question 13

Temperature at which organisms function best is determined by:

Answer 13

Proteins, primarily because enzymes control all cells activities and only function within properly within narrow temperature ranges

Question 14

Optimal temperature

Answer 14

A temperature at which the enzyme functions at its highest rate. If a cell and its enzymes are exposed to a temperature higher than optimal, the 3D structure of the enzyme may be altered. If the structure is altered it may no longer attach to the appropriate substrate.

Question 15

Denaturation

Answer 15

Permanent alteration of the structure of an enzyme

Question 16

Thermolabile

Answer 16

Enzymes that are unable to tolerate even slight increases in temperature above their optimal without denaturation

Question 17

Thermostable

Answer 17

Enzymes that can tolerate moderate temperature increases without permanent damage

Question 18

Temperatures below optimum

Answer 18

Usually do not cause changes in the structure but rather affect the dynamics of their actions.

• Slower growth
• Slow diffusion on molecules thus slowing enzymatic activity
• Reversible when temperatures are returned to normal.
• freezing may preserve bacteria by slowing down metabolism, and therefore the need for nutrients, until they are later thawed

Question 19

Psychrophiles

Answer 19

Grow from -5C-20C
Includes organisms that grow in cold water, soils, and in the refrigerator.
Ex. Listeria (a human pathogen that can grow well at these temperatures)

Question 20

Mesophiles, 2 subdivisions

Answer 20

Grow from 20C-45C
Grow well within the temperatures at which humans and other living things live, and at which many foods are held for consumption
Divided into:
-Room temp mesophiles: Growing between 20C and 35C
-Body temp mesophiles: Found on warm blooded animals as mutualistic normal flora or pathogens, prefer temps from 35C-45C

Question 21

Thermophiles

Answer 21

Have very stable thermostable enzymes and thus flourish in hot environments
45C and higher
Found in hot tubs, buffet warmers, air conditioners
Includes human pathogen legionella pneumophila

Question 22

Aerobe

Answer 22

• Need oxygen
• Glycolysis->krebs->ETC
• Need O2 to catch electrons at end of the ETC which makes water
• In all living systems: O2+H2o—>H2O2 (toxic)
• Any organism that lives in an aerobic environment must be able to breakdown hydrogen peroxide

Question 23

Anaerobes

Answer 23

• Dont require oxygen
• Glycolysis-> fermentation
• Since anaerobes are not competing with aerobes for nutrients, efficiency is not important due to virtually unlimited resources

Question 24

Obligate anaerobes

Answer 24

Do not posses the ability to breakdown hydrogen peroxide and will self destruct in the presence of O2. Many of these organisms from endospores to tolerate oxygenated conditions until the environment becomes more favorable

Question 25

Aerotolerant anaerobes

Answer 25

Are able to survive in the presence of oxygen although they do not used oxygen in their metabolism.

Question 26

Facultative

Answer 26

Flexible based on oxygen needs and tolerance. -posses all of the aerobic enzyme systems (glycolysis, krebs etc) as well as fermentative enzymes, and typically produce both catalase and superoxide dismutase. Gives these organisms the ability to function well in any type of environment, both oxygen rich and oxygen free. However, tend to grow better aerobically than anaerobically bc produce more ATP aerobically. Ex. E. Coli

Question 27

Microaerophiles

Answer 27

Small air loving bacteria which posses unique and various enzyme systems which function most efficiently in an atmosphere which has reduced oxygen levels as well as increased CO2. (this is the proportion of gases in healthy living tissues) -most live within other organisms, either mutualistic or as pathogens Ex. Nisseria and strepococcus

Question 28

Gas-Pak

Answer 28

- Most widely used anaerobic incubation system in labs and small medical settings. - Large, plastic, sealable jar, in which plates are placed. Before sealing a foil packet (CO2 and H2 generator) is activated the H2 produced in the jar will combine with free O2 to form H2O (condensation) on the inside of the jar effectively removing O2. - A methylene blue indicator strip is added. It will convert from the oxidized state (blue) to the reduced state (colorless) when free O2 has been removed. - Co2 released by the generator encourages the growth of microaerophiles within the jar, even though there is less oxygen than they prefer.

Question 29

Thioglycollate broth

Answer 29

-Has several unique features to permit the growth of anaerobes and microaerophiles: 1. Dehydrated broth is mixed with water and heat sterilized. The heat drives out oxygen and the tightened caps prevent it from reentering. * broth contains agar - to slow the diffusion of oxygen into the broth from surrounding air. Active ingredient: Na thioglycollate brings to the small amount of Oxygen in the air space as it re enters the broth upon cooling. * resazurin - oxidation/reduction indicator. becomes red in the presence of oxygen in the top laters while remaining colorless in the anaerobic bottom two thirds of the broth.

Question 30

Which two ingredients ensure that oxygen is captured within the top layers of the broth? What are the other functions of each one?

Answer 30

Na thioglycollate, and agar Agar slows down diffusion of oxygen, and Na binds to the oxygen preventing it from traveling to the bottom

Question 31

Thioglycollate broth gradient

Answer 31

Aerobic conditions in the top, red area; microaerophilic conditions in the bottom of the red area and the top of the clear area; and anaerobic conditions in the bottom, clear area.

Question 32

Isotonic

Answer 32

Cells environment has same concentration of solutes as inside of cell

Question 33

Hypotonic

Answer 33

Cells environment has lower concentration of solutes than inside of cell -will cause cell most animal cells to swell and burst. but plant, fungal, and bacterial cells which have cell walls the increased fullness actually increases the cells efficiency.

Question 34

Hypertonic

Answer 34

Cells environment has higher concentration of solutes than inside of the cell This situation causes water to move out of the cell. This is detrimental to all cell types, since the presence of rigid cell walls cannot protect against dehydration and death due to massive water loss.

Question 35

Halophiles

Answer 35

-can live in highly salty environments (hypertonic solutions) without being adversely affected.

Question 36

Halotolerant

Answer 36

Organisms which tolerate some additional salt in the environment but don't require salt. ex. staph, which makes up normal skin flora

Question 37

Why are fungi more resistant to osmotic pressure from sugar solutions?

Answer 37

Fungi are very metabolic and can digest/use the sugar in their environment to make it more isotonic which thus prevents them from drying out and dying.

Question 38

Antiseptics

Answer 38

Chemicals used on living surfaces such as skin to kill or inhibit the growth of bacteria

Question 39

Disinfectants

Answer 39

Chemicals used on inanimate objects to kill or inhibit the growth of bacteria

Question 40

Antibiotics

Answer 40

Chemicals used in the treatment of disease rather than just to inhibit or kill any bacteria present. Most are taken internally and have a systemic effect on the person taking the drug.

Question 41

Bacteriocidal

Answer 41

Kill bacteria outright

Question 42

Bacteriostatic

Answer 42

Prevent the reproduction of bacteria

Question 43

Narrow spectrum antibiotics

Answer 43

Action is specific for one genus or category of bacteria

Question 44

Broad spectrum antibiotics

Answer 44

Broad spectrum drugs affect a wide variety of organisms, both pathogens and normal flora, while narrow spectrum drugs may allow the pinpoint killing of specific species without affecting beneficial organisms.

Question 45

Ways in which antibiotics can affect bacteria

Answer 45

- Cell wall interference - Block protein synthesis - Block metabolites - Inhibit nucleic acid synthesis

Question 46

Problem with using antimicrobials against bacteria

Answer 46

Can cause bacteria to develop resistance to the agents we use to kill them

Question 47

Kirby bayer method | Name of the agar medium?

Answer 47

Involves the swabbing of a hinton muller agar medium for confluent growth with a selected organism, and the subsequent placement of disks soaked in antimicrobial agents on the surface. The agent will diffuse in all directions into the agar from the disk, with the highest concentration found directly under the disk, and the lowest concentrations further from the disk.

Question 48

Zone of inhibition

Answer 48

Absence of growth around the disk. Absence of growth means that the organism swabbed on the plate was unaffected by the agent

Question 49

Other factors that affect the zone of inhibition (other than sensitivity)

Answer 49

- Solubility of the drug in water - Size of the drug molecule - Time or temperature of incubation - Inactivation of the drug by substances in the medium - Concentration of drug on the disk

Question 50

UV radiation, describe wavelength, and energy

Answer 50

- Excite molecules to the point of disruption, useful only on surfaces due to its inability to penetrate glass, plastic or any other solid or liquid. - Long wavelength, low energy (why they don't penetrate) - UV light causes thymine dimers within the DNA of the cell, when this occurs adjacent thymine nucleotides bind to each other, rather than adenine. Distortion of the DNA molecule will occur, and the cell may be unable to replicate or transcribe its DNA properly. - Bacterial cells are sensitive to the effects of UV light, while spores are somewhat resistant. Viruses will be destroyed. Thus UV radiation is cheap and easy to use to disinfect areas. (sanitizes)

Question 51

Gamma radiation, describe wavelength and energy

Answer 51

- Much smaller wavelengths but much higher energy - Can penetrate well through glass, paper, and large volumes of liquid. - Cause the formation of free radicals - Ionizes ALL molecules rather than just DNA like UV light - Can be used to sterilize already packaged food, but is expensive and very dangerous (requires thick lead lined walls)

Question 52

Enriched media

Answer 52

Have extra nutrients added above and beyond the normal ingredients found in tryptic soy agar (TSA) or nutrient agar media - Often used to grow bacteria that have special needs ex. blood agar, chocolate agar, milk in litmus milk tubes, manure and brain heart infusion.

Question 53

Selective media

Answer 53

Media which have ingredients to inhibit the growth of one population of organisms while allowing other groups to grow -Includes salts, dyes, and antibiotics

Question 54

Differential media

Answer 54

Allow the bacteria growing on them to be differentiated into two or more groups based on the characteristics of the growth on the medium. -Includes agents such as: sugars, and pH indicators to differentiate fermenters from non fermenters and red blood cells to determine the ability of organisms to lyse the red blood cells.

Question 55

Blood agar | Is it enriched, selective, differential and what for?

Answer 55

Enriched: -Whole blood added, which supplies additional proteins, solutes, iron, and other nutrients. Differential for: -Hemolysis production, ingredient: RBC's.

Question 56

Mannitol Salt Agar (MSA) | Is it enriched, selective, differential and what for?

Answer 56

Has high salt concentration which inhibits most organisms other than staph. - Selective for staph, ingredient salt - Differential for: Mannitol fermentation, ingredient mannitol and pH indicator

Question 57

Phenylethyl Alcohol Agar (PEA) | Is it enriched, selective, differential and what for?

Answer 57

Phenylethyl is a strong inhibitory agent for gram negatives | -Selective for Gr+, ingredient: alcohol

Question 58

Eosin Methylene Blue Agar (EMB) | Is it enriched, selective, differential and what for?

Answer 58

Contains eosin (a pink dye) and methylene blue ( a blue dye). Inhibits Gr+ bacteria. Lactose is included. - Selective for Gr-, ingredient: dye - Differential for lactose ferm, and e.coli, ingredients: Lactose and pH indicator *e.Coli will appear greenish gold

Question 59

Maconkey's Agar (MAC) Is it enriched, selective, differential and what for? What does purple color indicate? Brownish color?

Answer 59

Uses crystal violet, and bile salts to inhibit the growth of Gr+ organisms. - Selective for: Gr-, ingredients: crystal violet and bile salts - Differential for: Lactose fermentation ingredient: pH indicator ``` Purple = lactose fermentor Browinish/rose = non lactose fermentor ```

Question 60

What technique could you do to find out of there is contamination present and what would you expect to see after incubation confirming contamination?

Answer 60

Isolation streak technique | Result showing multiple colony types or appearances would confirm contamination

Question 61

What issues arise with the use of gamma irradiation?

Answer 61

- expensive - dangerous - requires a lot of space - people are afraid of irradiated food

Question 62

What reagent could be used in place of resazurin

Answer 62

methylene blue

Question 63

What two media are differential for lactose ferm?

Answer 63

EMB and MAC

Question 64

A PEA plate is selective for..?

Answer 64

Gr+, ingredient: alcohol

Question 65

Genus of psychrophilic bacteria which is pathogenic, especially for pregnant women

Answer 65

Listeria

Question 66

Genus of thermophilic bacteria which is pathogenic, associated with a convention in 1976

Answer 66

Legionella

Question 67

Name of the medium used to test antibiotics effectiveness against bacteria

Answer 67

``` Muller Hinton (Kirby bauer test) ```

Question 68

Name for bacterial cells which have been treated to make them ready to absorb DNA fragments

Answer 68

Competent

Question 69

Thioglycollate broth with an Anaerobe

Answer 69

Growth only in the uncolored anaerobic zone (bottom)

Question 70

Thioglycollate broth with an Aerobe

Answer 70

Growth only in the pink aerobic zone located at the top

Question 71

Thioglycollate broth with a Microaerophile

Answer 71

Growth at the top with a stalagmite that grows towards the bottom

Question 72

Thioglycollate broth with a faculative organism

Answer 72

Growth all throughout the broth

Question 73

Drawback to using thioglycollate broths

Answer 73

Cultures must be isolated and pure before being placed in the tube. Isolation streaks from a wound or throat cannot be incubated in this manner; instead and two step procedure of isolation in a Gas-Pak or special incubator followed by thioglycollate tubes must be used, increasing time needed to ID pathogen Any pathogens requiring extra nutrients will not grow well

Question 74

Category of oxygen need in which the organisms have the MOST energy producing enzymes

Answer 74

Facultative

Question 75

Method to determine whether the bacteria in a zone of inhibition around a drug disk are killed or simply growth inhibited

Answer 75

Swab in the zone of inhibition and subculture in another medium. If the bacteria grow it could be inferred that the drug is bacteriostatic but if the bacteria don't grow then the drug is bactericidal

Question 76

Why do doctors not routinely perform sensitivity tests?

Answer 76

- Wanna get medicine into an individual as soon as possible to prevent them from infecting other and/or suffering any longer - Most insurance companies won't pay for sensitivity testing unless 2 other antibiotics have failed - Doctors want to keep their patients happy and coming back and know that they won't come back if they don't walk out with a prescription.

Question 77

EMB plate | differential, enriched, or selective for? colors?

Answer 77

Selective for Gr-, ingredient methylene blue Differential for Lactose ferm, and E.Coli, incredients lactose and pH indicator E.Coli = greenish gold

Question 78

MAC differential, enriched, or selective for? Color appearance

Answer 78

Selective for Gr-, ingredients: crystal violet and bile salts Differential for: lactose ferm, ingredients: pH indicator Purple: lactose ferm Brown: Not lactose ferm