Lab Test 2 Flashcards
Why is the isolation of organisms necessary?
- 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
Pure culture
A single species (or strain) of bacteria growing in a culture; essentially a colony of cloned cells which are all identical
Isolation streak technique
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)
What are 2 ways that the number of bacteria on your loop are reduced during the isolation streak technique?
- Flaming
- Rubbing the loop on the agar
Subculturing
The process of aseptically transferring a culture from one culture medium to another
What is the purpose of subculturing?
- 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.
Important things to remember during the subculturing technique:
-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.
Five characteristics observed from a slant culture:
-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
3 characteristics observed from a broth culture
-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.
5 characteristics of isolated colonies
- 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
Characteristics of mycobacterium
Very waxy! will form large pellicle, irregular shaped colony
What factors could affect the normal pattern and cause uncharacteristic growth patterns in a species
- 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
Temperature at which organisms function best is determined by:
Proteins, primarily because enzymes control all cells activities and only function within properly within narrow temperature ranges
Optimal temperature
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.
Denaturation
Permanent alteration of the structure of an enzyme
Thermolabile
Enzymes that are unable to tolerate even slight increases in temperature above their optimal without denaturation
Thermostable
Enzymes that can tolerate moderate temperature increases without permanent damage
Temperatures below optimum
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
Psychrophiles
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)
Mesophiles, 2 subdivisions
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
Thermophiles
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
Aerobe
- 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
Anaerobes
- Dont require oxygen
- Glycolysis-> fermentation
- Since anaerobes are not competing with aerobes for nutrients, efficiency is not important due to virtually unlimited resources
Obligate anaerobes
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
Aerotolerant anaerobes
Are able to survive in the presence of oxygen although they do not used oxygen in their metabolism.
Facultative
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
Microaerophiles
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
Gas-Pak
- 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.
Thioglycollate broth
-Has several unique features to permit the growth of anaerobes and microaerophiles:
- 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.
Which two ingredients ensure that oxygen is captured within the top layers of the broth? What are the other functions of each one?
Na thioglycollate, and agar
Agar slows down diffusion of oxygen, and Na binds to the oxygen preventing it from traveling to the bottom
Thioglycollate broth gradient
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.
Isotonic
Cells environment has same concentration of solutes as inside of cell
Hypotonic
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.
Hypertonic
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.
Halophiles
-can live in highly salty environments (hypertonic solutions) without being adversely affected.
Halotolerant
Organisms which tolerate some additional salt in the environment but don’t require salt.
ex. staph, which makes up normal skin flora
Why are fungi more resistant to osmotic pressure from sugar solutions?
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.
Antiseptics
Chemicals used on living surfaces such as skin to kill or inhibit the growth of bacteria
Disinfectants
Chemicals used on inanimate objects to kill or inhibit the growth of bacteria
Antibiotics
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.
Bacteriocidal
Kill bacteria outright
Bacteriostatic
Prevent the reproduction of bacteria
Narrow spectrum antibiotics
Action is specific for one genus or category of bacteria
Broad spectrum antibiotics
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.
Ways in which antibiotics can affect bacteria
- Cell wall interference
- Block protein synthesis
- Block metabolites
- Inhibit nucleic acid synthesis
Problem with using antimicrobials against bacteria
Can cause bacteria to develop resistance to the agents we use to kill them
Kirby bayer method
Name of the agar medium?
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.
Zone of inhibition
Absence of growth around the disk. Absence of growth means that the organism swabbed on the plate was unaffected by the agent
Other factors that affect the zone of inhibition (other than sensitivity)
- 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
UV radiation, describe wavelength, and energy
- 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)
Gamma radiation, describe wavelength and energy
- 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)
Enriched media
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.
Selective media
Media which have ingredients to inhibit the growth of one population of organisms while allowing other groups to grow
-Includes salts, dyes, and antibiotics
Differential media
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.
Blood agar
Is it enriched, selective, differential and what for?
Enriched:
-Whole blood added, which supplies additional proteins, solutes, iron, and other nutrients.
Differential for:
-Hemolysis production, ingredient: RBC’s.
Mannitol Salt Agar (MSA)
Is it enriched, selective, differential and what for?
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
Phenylethyl Alcohol Agar (PEA)
Is it enriched, selective, differential and what for?
Phenylethyl is a strong inhibitory agent for gram negatives
-Selective for Gr+, ingredient: alcohol
Eosin Methylene Blue Agar (EMB)
Is it enriched, selective, differential and what for?
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
Maconkey’s Agar (MAC)
Is it enriched, selective, differential and what for?
What does purple color indicate? Brownish color?
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
What technique could you do to find out of there is contamination present and what would you expect to see after incubation confirming contamination?
Isolation streak technique
Result showing multiple colony types or appearances would confirm contamination
What issues arise with the use of gamma irradiation?
- expensive
- dangerous
- requires a lot of space
- people are afraid of irradiated food
What reagent could be used in place of resazurin
methylene blue
What two media are differential for lactose ferm?
EMB and MAC
A PEA plate is selective for..?
Gr+, ingredient: alcohol
Genus of psychrophilic bacteria which is pathogenic, especially for pregnant women
Listeria
Genus of thermophilic bacteria which is pathogenic, associated with a convention in 1976
Legionella
Name of the medium used to test antibiotics effectiveness against bacteria
Muller Hinton (Kirby bauer test)
Name for bacterial cells which have been treated to make them ready to absorb DNA fragments
Competent
Thioglycollate broth with an Anaerobe
Growth only in the uncolored anaerobic zone (bottom)
Thioglycollate broth with an Aerobe
Growth only in the pink aerobic zone located at the top
Thioglycollate broth with a Microaerophile
Growth at the top with a stalagmite that grows towards the bottom
Thioglycollate broth with a faculative organism
Growth all throughout the broth
Drawback to using thioglycollate broths
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
Category of oxygen need in which the organisms have the MOST energy producing enzymes
Facultative
Method to determine whether the bacteria in a zone of inhibition around a drug disk are killed or simply growth inhibited
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
Why do doctors not routinely perform sensitivity tests?
- 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.
EMB plate
differential, enriched, or selective for? colors?
Selective for Gr-, ingredient methylene blue
Differential for Lactose ferm, and E.Coli, incredients lactose and pH indicator
E.Coli = greenish gold
MAC
differential, enriched, or selective for?
Color appearance
Selective for Gr-, ingredients: crystal violet and bile salts
Differential for: lactose ferm, ingredients: pH indicator
Purple: lactose ferm
Brown: Not lactose ferm
