First lab Flashcards
(206 cards)
1
Q
Two organizations that put forth procedures and techniques to help prevent the spread of infectious diseases?
A
(CDC) Center for Disease Control and Prevention and (WHO) World Health Organization
2
Q
A huge number of potentially preventable deaths occurred during the_______war.
A
Civil war
3
Q
How do we disinfect our work place in the Microbiology lab?
A
We decontaminate the bench before and after lab period. Spray the desk with disinfectant, take a towel and distribute it over the table top and let it continue to dry on its own.
4
Q
How did Dr. Lister (1800’s) disinfect his operating room?
A
With a diluted solution of carbolic acid (Phenol).
5
Q
Do you place hazardous, infectious materials in the sink? This also includes solid material (this includes paper towel)
A
NO
6
Q
How do you clean up spills of microbial cultures?
A
Cover the contaminated area w/ a paper towel that is soaked throughly w/ disinfectant. Let it remain on spill for ten minutes, then use paper towels to soak up spill. Towel goes in biohazard waste bag. Wash hands immediately afterwards.
7
Q
What do you do w/ broken glass slides?
A
Don’t pick up w/ bare hands, sweep up the broken glass and place them in the specified container.
8
Q
How do you dispose of glass slides and coverslips?
A
Wrap them in a paper towel and dispose of them in the biohazard container. Don’t discard demonstration slides.
9
Q
Contaminated waste goes in biohazard bags, so where do non-biohazard waste belong?
A
Can be disposed of in appropriate containers, gloves can go in regular trash cans.
10
Q
Where does inoculated media go?
A
To the 37 degree celsius incubator or room temperature storage tub, located in minilab (S213). We are responsible for proper disposal of all test tubes/petridishes in biohazard bags.
11
Q
For short term storage of inoculated media?
A
Black refrigerators
12
Q
When should you wash your hands thoroughly with soap and water?
A
Before class, after class, or when you spill something.
13
Q
What are some good aseptic techniques to practice before class?
A
–Tie hair back –Wear a clean lab coat and gloves at all times –Decontaminate work bench before/after class –Wash hands throughly before/after class w/ soap/water
14
Q
What are the five steps to hand washing?
A
Wet hands with water Apply hand wash (soap) Lather and wash for AT LEAST 15 seconds Rinse both sides of hands with water Dry hands and shut off faucet with towel
15
Q
Two big groups of organisms found on your hands?
A
Resident and transient flora are on your hands.
16
Q
Preventing pathogens from being transmitted from recognized and unrecognized sources?
A
Standard precautions includes the use of; hand washing, appropriate personal protective equipment such as gloves, gowns, masks, whenever touching or exposure to patients’ body fluids is anticipated.
17
Q
If you are in an emergency situation, where hand washing isn’t possible what should you do?
A
Use an alcohol-based hand sanitizer.
18
Q
What are nonsocomial disease and what is the number one way to prevent the spread of nonsocomial infection?
A
They are acquired in hospitals/healthcare facilities. To be classified as a nosocomial infection, the patient must have been admitted for reasons other than the infection. Some patients acquire nosocomial infections by interacting with other patients. Hospital staff can significantly reduce the number of cases with regular hand washing. They should also wear protective garments and gloves when working with patients.
19
Q
What is an inoculating loop?
A
Allows the microbiologist to handle specimens in an aseptic manner. Has an aluminum handle and thin wire filament that rapidly heats up and cools down.
20
Q
What does the Bunsen Burner and Incinerator do?
A
Bunsen Burner/Incinerator-connected by tubing to gas jet, ignited, and the flame is then used to sterilized the inoculating loop.
21
Q
What should you do or not do after sterilizing your inoculating loop with the bunsen burner?
A
By inserting the inoculating loop in the hottest part of the flame you are using aseptic technique to sterilize it. Let the wire cool down for a few seconds before transferring your specimen from one medium to another. Do not wave in the air or blow on the inoculating loop.
22
Q
What do sterile media consist of?
A
Nutrients for growing bacteria
23
Q
What is the in vitro environment in which bacterial cells grow?
A
Medium
24
Q
What is Agar?
A
A nutritionally inert substance that gives media its solid consistency. Must be heated to boiling to dissolve but will solidify at 40C. Can be allowed to solidify in tubes in an upright position, resulting in media termed stabs or deeps or in a slanted position producing slants w/ more surface area than stabs. Agar can also be poured to shallow dishes called Petri dishes to produce plates.
25
Definition of Sterile?
Material is free of all living forms.
26
What does the Autoclave do?
An autoclave is a pressure chamber used to sterilize equipment and supplies by subjecting them to high pressure saturated steam (moist heat) at 121 °C (249°F) for around 15–20 minutes depending on the size of the load and the contents. Kills even the most resistant endospores.
27
How should you label your media?
Use a sharpie to write your initials, the date, the type of medium, and the inoculum (specimen you are culturing) on the class of culture tube (never on lid) or the bottom side (w/agar) of the petri dish.
28
When working w/ media where should you place it at your work space?
Towards the middle, away from notebooks/books. Tubes should be placed in a test tube rack and petri dishes should be placed on top of the rack and secured w/ masking tape.
29
List the tube media.
TSB (Tryptic Soy Broth) TSD (Tryptic Soy Deep) TSS (Tryptic Soy Slant)
30
List plate media
TSA (Tryptic Soy Agar) TSP (Tryptic Soy Plate)
31
Aseptic vs. Sterile
Aseptic-free from contamination caused by harmful bacteria, viruses, or other microorganisms. (of surgical practice) aiming at the complete exclusion of harmful microorganisms. (of a wound, instrument, or dressing) surgically sterile or sterilized. Sterile-free from bacteria or other living microorganisms; totally clean.
32
How many Microbes are estimated by Scientist to be on earth?
5x10(30)
33
Microbes are ubiquitous (exist everywhere)? T or F ?
True
34
Can you explain why microbes are widespread?
1.) Their small sizes allow for easy dissemination by air and water. 2.) Demonstrate incredible versatility and flexibility w/ regards to their metabolism. 3.) Have a unique ability to tolerate extreme or unusual environments.
35
What do the microbes that live in oceanic and terrestrial environments do for earth?
The earth has limited quantities of carbon, nitrogen, and oxygen. Microbes are capable of recycling essential elements. Digesting the remains of once-living cells, returning basic chemicals back to the earth's biogeochemical cycles.
36
Describe high species diversity (low numbers of existing species).
1.) Found in niches that do not contain many selective pressures. 2.) In soils and sea water Species diversity is the number of different species that are represented in a given community.
37
Define species richness (present in high numbers).
1.) Ecosystems w/ strong selective pressures inhibit the growth of all but a few species. 2.) Bacteria that can survive or tolerate the disinfectants used in hospital room. Species richness is the number of different species represented in an ecological community, landscape or region.
38
In what type of environments would you expect to find a large species diversity of microbes?
In niches that do not contain many selective pressures, such as soils and sea water.
39
How is it that your not sick all the time?
Our body provides us w/ adequate protection against microbial infections.
40
Given that niches w/ low selective pressures tend to have high species diversity, how do you think microbes are able to find nutrients and prevent other species from utilizing these nutrients?
Microbes demonstrate versatility/flexibility w/ regards to their metabolism, and have the ability to tolerate extreme environments.
41
What characteristics define extremophile?
Characteristics of extremophiles. Extremophiles are organisms that live in extreme conditions of temperature, acidity, salinity, pressure, or toxin concentration. Most extremophiles are single-celled micro-organisms belonging to two domains of life – bacteria and archaea.
42
Describe an extremophile.
Extremophiles are organisms that have been discovered on earth that survive in environments that were once thought not to be able to sustain life.
43
Why do the discoveries of extremophiles lead us to hypothesize that microbes may live elsewhere than Earth?
Possibility that life forms elsewhere might be a lot like the extremophiles that live in similar harsh conditions here on Earth. Microbes could live in the ice deep within comets, frozen there for eons until a collision with another planet or moon delivered them to a new home.
44
Synthetic media (defined media)?
Nutrient preparations used for microbial growth, the type of medium, exact composition and quantity of each nutrient is known. Used for fastidious microorganisms.
45
Complex or Chemically undefined media?
These types of media contain the same elements as defined media, but the amounts of each particular nutrient may vary from recipe to recipe.
46
What is Agar?
A polysaccharide solidifying agent derived from marine algae. Few organisms degrade it, it does not melt until its temperature reaches 95C and after being melted, it remains in a liquid state until it has cooled to about 40C.
47
What is an Agar Slant?
Liquefied medium that contains Agar and can be poured into a test tube or petri plate. The medium cools down and solidify's at a slant.
48
What is Agar Deep?
Liquefied medium is allowed to solidify w/ the tube remaining upright.
49
What is an Agar Pour?
Medium that is poured into a large test tube and is intended for pouring into a petri plate at a later date.
50
What is an Agar Plate?
Liquefied medium poured into a petri plate in order to create a solid surface for growing bacteria.
51
Describe Dry-heat sterilization.
Used mostly for glassware, consist of a hot air oven at 160-170C for 2 hrs.
52
Describe Filtration
Used for media additives that cannot w/stand high heat sterilization methods. Liquid is passed through a filter that traps microorganisms while allowing the liquid to continue through the filter into a sterile container.
53
Describe Ultraviolet radiation.
Used in hospitals to sterilize the air or surfaces in a room.
54
Describe ethylene oxide.
A gas used to sterilize heat-sensitive materials, such as plastic petri dishes, cotton packing, and syringes.
55
What is the most important thing to remember about sterilization?
Materials cannot be 97% sterile-they are either sterile or not sterile.
56
List sterilization methods.
Autoclave, moist heat, dry-heat, filtration, ultraviolet radiation, ethylene oxide gas.
57
What is contamination?
The act of contaminating, or of making something impure or unsuitable by contact with something unclean, bad, etc. You can contaminate by your hands, the air you exhale, or inanimate objects. Microorganisms are everywhere.
58
What is aseptic technique?
Preserving the purity of objects.
59
How do you sterilize the inoculating loop?
Use the Bunsen burner to sterilize the inoculating needle by passing the base (closest to handle) through a flame to kill microorganisms. It must glow orange. Let the loop cool for several seconds. Do not set the loop down on the bench, don't blow on it, do not touch it w/ your fingers to see if its still hot.
60
Why mustn't you shake a tube of broth media?
The caps of the broth media have holes in them to allow for exchange of gasses. Shaking them will spill the media everywhere.
61
What to do w/ a cap when you take it off a test tube?
Caps remain on flask/tube any time the flask/tube is not in use. When you remove a cap you flame the mouth of the tube. The lip should be held at an angle to reduce the possibility that organisms in the air might fall into the tube. Never set the tube on the bench, remove the cap w/ the little finger of the hand holding the inoculating loop.
62
Describe pure culture.
Simply a culture of one type of organism.
63
What is inoculation (subculturing similar to inoculation)?
Microbiologist use this to transfer a pure culture of an organism. A tiny amount of microorganism is removed from one medium using an inoculating loop or an inoculating needle, and transferred or inoculated into or onto fresh medium.
64
Removing organisms from a liquid culture?
1.) Always grasp tubes by the glass portion, not the cap. 2.) Settling of the bacteria in broth tubes may have occurred. 3.) Gently shake or vortex broth tubes to resuspend organisms. Do not invert the tubes to mix the organisms (remember holes in the cap). 4.) Sterilize the inoculating loop, let it cool (10secs). 5.) Remove cap of the culture tube containing the inoculum w/ your little finger of the same hand that is holding the inoculating loop and hold the cap next to your palm. Flame the lip of the tube, two-three times, holding tube at an angle to minimize contamination. 6.) Stick cooled inoculating loop into the liquid bacterial culture. You should see liquid in the loop. 7.) Flame the lip of the tube containing the liquid culture. Replace the cap and return tube to the rack. 8.) Sterilize the loop.
65
What happens if your inoculating loop is to hot?
You'll hear a hissing sound when you put it in liquid broth, which indicates that you are creating aerosols (steam), and you are also killing bacteria.
66
Removing organisms from slant culture?
1.) Sterilize loop and lip of test tube 2.) Insert loop into tube, scrape some of bacteria growing on surface of the medium using loop. Do not break the surface of the agar. A small amount of visible organism will suffice. 3.) Flame lip of the tube and transfer inoculum to fresh medium. 4.) Sterilize loop.
67
Removing organism from an Agar Plate Culture?
1.)Agar is contained in the bottom portion of the plate. Place the petri dish w/ the lid of the dish (big side) on the bench top. 2.) Sterilize loop. 3.) Pick up smaller bottom-side of petri dish that is filed w/ the agar w/ the other hand. 4.)Use loop to gently scrape a small amount of growth from the agar w/out gouging the agar. Tiny amount of organism is needed. 5.) Replace the bottom of petri dish on the lid and transfer the inoculum to fresh medium. 6.) Sterilize loop.
68
Why do we set the petridish upside down?
If you left it right-side up condensation would appear on the inside of the top lid and would drown the bacteria.
69
Inoculating broth medium?
Remove cap, flame lip of tube. Dip loop into broth, gently move loop back/forth to transfer organism from loop to the new medium. Flame neck of tube and replace cap.
70
Inoculating an agar slant?
Stab the loop down to the butt of the tube, bring loop back up the slant w/ a zig-zag motion, smear the rest of the inoculum on the slant portion of the agar. Flame neck of tube, replace cap.
71
Inoculating an agar deep?
Remove cap, sterilize lip of test tube. Stab loop 3/4 of the way to bottom of the deep, carefully w/draw loop. Do not need to move loop around b/c the mechanical action of stabbing the medium causes organisms to be transferred to the agar deep from the loop. Flame neck of tube, and cap it.
72
Inoculating an agar plate?
Place petri dish w/ the top of the dish (big side) on the bench top. While holding loop w/ the inoculum in one hand, pick up the smaller bottom side of the petri dish that is filled w/ the agar. With other hand gently streak the loop along the surface of the agar to transfer organisms. Without Breaking surface of medium.
73
Spot inoculation? (agar plate)
A concentrated area of organism about the size of a quarter is placed on the plate.
74
Streak-for-isolation-technique? (agar plate)
Bacteria are diluted across the surface of the medium in an attempt to isolate individual colonies of bacteria.
75
Why should a microbiologist wait until a liquid medium containing medium agar has cooled to 45C before adding living organisms?
The heat from the liquid could kill organisms. At this temperature the necessary nutrients can be added and it will have solidified.
76
Explain why you sterilize an inoculating loop? Before dipping into a tube?
Sterilizing the inoculating loop prevents contamination of the pure culture. And to sterilize after inoculation to kill/sterilize any microorganisms on the loop.
77
Explain why tubes should be kept as close to parallel as possible to the bench top while performing any transfers of inoculations?
To prevent airborne particles from contaminating the inside of the tube.
78
Most microbes cannot be grown by inoculating them into sterile water. Why? What must be provided for organisms to grow?
A standard carbon source is glucose, and nitrogen is often provided by peptones (partially digested proteins), or inorganic salts. Minerals and vitamins may also be provided, according to the growth requirements of the bacteria. Combinations of chemicals (buffers) may be used to keep the pH stable. Measured amounts of the concentrates are added to water, and dissolved to reconstitute the media.
79
What is a fomite?
An inanimate object or substance, such as clothing, furniture, or soap, that is capable of transmitting infectious organisms from one individual to another.
80
What is symbiosis?
Interaction between two different organisms living in close physical association, typically to the advantage of both.
81
What is a mixed culture?
Many types of microorganisms.
82
Pure culture?
Growth of only one type of microorganism in media w/out the presence of any other organism in that culture.
83
How do bacteria reproduce?
Binary fission, one mother cell, when provided w/ the proper environment, will divide into two identical daughter cells. Each daughter cell will then divide into two daughter cells and when this continuous division takes place on solid media, all of these identical cells will begin to form a colony on the medium consisting of identical cells.
84
What is streak-plate technique?
Using your inoculating loop to streak an aliquot (portion) of the original mixed culture on a petridish of medium such as TSA (tryptic soy agar) and then dilute the original inoculum to allow individual cells to rest on the medium and grow. (Separating the organisms in the sample and producing individual colonies of bacteria).
85
Tryptic Soy Agar (TSA)?
Complex medium that contains digests of casein and soybean meal as sources of carbon, and energy, glucose as an energy source, and various salts. Allows for the growth of a wide variety of microorganisms from both clinical and nonclinical specimens. During incubation of the TSA, individual cells multiply by binary fission and produce separate isolated colonies, each colony consisting of only one type of bacterium. As long as the colony's aren't touching each other, each colony will consist of daughter cells which came from one mother cell.
86
Stock plate?
Each individual of colony can be transferred to fresh culture medium and the resulting growth produces many colonies of one species of bacteria or pure culture. This pure culture can be used as an inoculum source for further study of that species of bacteria.
87
Quadrant method?
Helps you visualize the boundaries of where you are working on the petridish.
88
Why do microbiologist use the streak-plate method, whats the purpose?
Going from a mixed culture to a pure culture that will separate the organisms in the sample and produce individual colonies of bacteria.
89
Why do you flame the loop between each streak?
To sterilize the loop and prevent contamination.
90
You streak a culture and find that the colonies in Quadrant lll are larger in diameter than the colonies in quadrant l. What would account for this?
Some where in the process of streaking your culture you contaminated it. Through access of airborne microorganisms or your sterilization method.
91
Can a pure culture containing a single type of microbe be prepared from a culture w/ a mixture of cells?
Yes, one such technique is streak plate in which you use your inoculating loop to streak a portion of the original mixed culture on a petri dish of mediums such as TSA and the dilute the original inoculum to allow individual cells to rest on the medium and grow.
92
Studying the results of your streak plates, how could you determine if a culture that you choose to isolate is a pure culture?
A pure culture will only be one type of microorganism in the media w/out the presence of any other organism in that culture.
93
Describe Standard Plate Count.
Used to determine the concentration of bacteria in a liquid medium. Achieved by doing a serial dilution and then doing a pour plate technique to produce isolated bacterial colonies on agar which can then be counted. Used to estimate the number of microbes present in a stock sample.
94
Serial Dilutions?
To reduce the number of microbes present. A small volume (aliquot) from each dilution tube is pipetted into 1ml of tryptic soy broth (TSB) which, is in turn poured into a sterile empty petri dish. Lastly, melted agar medium at 45C is poured into the petri plate, and the organism and medium mixture is swirled to ensure even distribution. The Agar liquid is allowed to solidify at 40-45C at room temperature, then the plates are inverted (upsides down) and incubated.
95
What is the advantage of using the pour plate technique over the steak plate technique?
It requires less skill.
96
Using the pour plate technique, you can observe colonies of bacteria growing not only on the surface as well as through out the medium. Why?
Colonies of bacteria will grow both on the agar surface and w/in the medium. Typical colonies form on the surface of the agar, but organisms immobilized in agar grow in three dimensions. These colonies do not resemble those that grow on the surface, and may even have a slightly different shape, but should be considered microbial colonies nonetheless.
97
What is a Quebec Colony Counter?
Used to estimate the number of microbes present in a stock sample.
98
What is common practice for the number of colonies to count on these plates?
30-300 Colonies.
99
Colony forming unit (CFU)?
Each colony is derived from a single microbe.
100
How to get the number of organisms in a stock culture?
(Pg. 44) Divide the number of CFU's by the dilution of the sample that was pipetted into the TSB. This dilution is the sum of the original dilutions of the stock culture plus any dilution incurred when the aliquot of the dilution was pipetted into the plate. Ex. 175 Colonies are present on a plate where the stock culture was diluted 1:1,000,000 (10 -6) and 1ml was plated: # colonies/(dilution factor)(volume plated)=175/(10-6)(1ml)= 1.75x10(8) CFU/ml
101
TFTC and TNTC
TFTC-to few to count TNTC-to numerous to count
102
Colony?
Thousands of new bacteria from a cell that had undergone binary fission.
103
Colony Morphology?
Studying the species of the bacteria to identify them.
104
Cultural Characteristics?
Genetic material of organism, whats typical of the genus and of a particular species. How they appear the same each time you observe that organism growing on that medium. -genetic make-up -type of media -environment conditions -concentration of atmosphere
105
The characteristics of bacterial growth on solid media?
Form-appearance of the entire colony Size-metric measurement of colony Margin-edge of the colony Texture-when manipulating colony w/ loop, does colony feel soft, granular/powdery, mucoid, or tenacious/clingy. Elevation-colony observed from the side. Surface appearance-creamy/smooth, shiny, dull, wet or dry. Pigment production-tan, beige, buff or white colonies are considered non-pigmented while pigmented bacteria have a variety of colors; yellow, gold, green, red, or pink. -----If pigment is produced and gives color not only to the colony but also to the agar, the pigment is hydrophilic and is water soluble. -----Some bacteria produce a hydrophobic pigment that only affects the colony itself and is termed water insoluble pigment.
106
Forms of bacteria?
(pg. 53) Punctiform, circular, spindle, concentric, irregular, swarming. Margin--Entire, undulate, scalloped, erose, lobate, spreading edge Elevation-falt, raised, subsurface, convex, pulvinate, umbonate.
107
(pg. 54) Bacterial growth of patterns in liquid media.
Uniform Turbidity-visible cloudiness evenly throughout the broth. Sediment of pellet-Accumlation of growth at bottom of the tube. Pellicle Formation-surface accumulation of growth.
108
Difference between antiseptics and disinfectants?
Antiseptics-Used on living tissues, such as a patients skin. Less toxic than disinfectants. Are often made to kill pathogens only, not all microbes. Disinfectants-Used on non-living surfaces, such as table tops.
109
What are the 8 categories of antiseptics/disinfectants.
1.) Phenol and Phenolics 2.) Alcohols 3.) Halogens 4.) Oxidizing Agents 5.) Surfactants 6.) Heavy metals 7.) Aldehydes 8.) Gaseous Agents
110
Phenol (disinfectant)
Disrupts plasma membrane and denatures proteins. Used to disinfect drains, cesspools, animal quarters. Apply wet and let air dry. (Lysol) Good against vegetative microbes, mycobacteria (leprosy and T.B.), fungi, and most viruses. Poor kill rate for endospores.
111
Alcohols (disinfectant and antiseptic)
Only ethyl and isopropyl alcohol is used to control microbial growth. Dissolves lipids of P.M. and denatures proteins. Diluted alcohol 70% can be used on skin to remove microbes. Inhibits growth of bacteria by dehydration, good for vegetative bacteria, fungi, and enveloped viruses but don't reliably kill bacterial endospores.
112
Halogens (disinfectant and antiseptic)
Iodine chlorine, bromide, and fluorine. Denature protein, example; the bleach used in your house. Kills most pathogenic bacteria and viruses.
113
Oxidizing Agents (antiseptic)
Peroxides, ozone, and peracetic acid. Causes denaturation of cellular proteins. Antiseptic for "closed" wounds. Used on hospital equipment.
114
Surfactants (antiseptics/disinfectant )
Soaps and detergents. Break through oily layer on skin (emulsification), once layer is disrupted the manual scrubbing helps remove debris and microbes. Disrupts the P.M. of microbes. Used in mouthwashes, general disinfectants. Very effective against Gram - bacteria.
115
Heavy Metals (antiseptic)
Not commonly used b/c they are toxic. Can cause pollution and bacteria develop a resistance against them. Combines and inactivates the functional groups of proteins. Destroy vegetative bacteria, fungi, algae, etc. Can't destroy endospores. Used as dressings that contain silver nitrate.
116
Aldehydes (disinfectant)
Used when you dissect a preserved specimen in HS biology. Formaldehyde! Powerful antimicrobials, disrupt functional groups of proteins and nucleic acids. Destroy vegetative bacteria, fungi, viruses, and bacterial endospore. Heat-sensitive medical instruments can be disinfected or sterilized w/ aldehydes.
117
Gaseous Agents (disinfectant)
Ethylene oxide gas is a sterilant that destroys all life forms. Denatures proteins/nucleic acids, needs a contact time of (4-18hrs). Used to disinfect fabrics, or sterilize materials in hospitals or dental offices.
118
What are Microbicidal agents?
Agents that result in microbial death.
119
What are Microbistatic agents?
Only temporarily inhibit microbial growth. Once this agent is removed the bacterial growth will resume.
120
Sterilants?
Destroy all microbes, their toxins and spores. To toxic to come into direct contact w/ humans or animals.
121
Antimicrobials (Chemotherapeutic agents)
Chemicals that decrease pathogens internally and may be naturally or synthetically produced. Antibiotics are a class of Chemotherapeutic agents produced naturally.
122
Phenol Coefficient: (pg. 63)
Robert Koch used this, method of measuring the antimicrobial effectiveness of disinfectants. Compares the kill rate of a given antimicrobial agent to the kill rate of a phenol.
123
Phenol Coefficient Formula (pg. 63)
Dilution rate for Disinfectant X/Dilution rate for phenol=175/50 =phenol coefficient of 3.5 Therefore the Phenol coefficient of Disinfectant X is 3.5 times stronger than phenol.
124
Selectively Toxic?
The must kill or inhibit microbial growth w/out causing permanent harm to the host.
125
Antibiosis
"Against life", the term antibiotic is derived from this word.
126
Antibiotic
Naturally produced substance made and secreted by a living microorganism that has microbicidal or microbistatic action action against other live microbes. Antibiotics have no effect on viruses b/c they are not alive.
127
Broad Spectrum
Antibiotics that are effective against a wide range of both gram positive and negative bacteria. Not the best choice b/c broad spectrum drugs can affect normal flora of the host and may allow for overgrowth of other organisms.
128
Narrow Spectrum
Antibiotics effective against only a restricted number of bacteria. Generally best to receive a drug this way with a narrow spectrum b/c side effects can be prevented. Not always the best b/c it takes several days to identify the agent of infection.
129
Kirby-Bauer test
Determining the susceptibility of an organism to an antibiotic. A Mueller-Hinton agar plate is swabbed w/ the organism in question and paper discs impregnated w/ antibiotics are placed on the surface of the agar, and then incubated for 24 hours. During incubation, the water soluble antibiotic diffuses through the agar, resulting in a concentration gradient of the antibiotic. As the antibiotic diffuses away from the antibiotic disc, the concentration of the antibiotic decreases, eventually becoming low enough to allow bacterial growth.
130
Minimal Inhibitory Concentration (MIC)
If a bacterial strain is susceptible to an antibiotic, no reproduction will occur until the antibiotic concentration drops below the MIC. The more susceptible a bacterial strain is to a particular antibiotic, the larger the clear area surrounding the antibiotic disc will be. This ring of no growth is called "zone of inhibition". The relationship of zone size to toxicity is not linear.
131
Determining Bacterial Susceptibility?
Measure the diameter of the zone of inhibition and compare to the zone diameter sizes found on standardized tables.
132
Contrast the terms disinfect and sterile.
Disinfect-clean (something) with a disinfectant in order to destroy bacteria. Sterile-free from bacteria or other living microorganisms; totally clean.
133
What is an antibiotic?
Naturally produced substance made/secreted by a living microorganism that has microbicidal or microbistatic action against other live microbes.
134
Are most antibiotics useful in treating viral infections? Why or why not?
They have no effect on viruses b/c viruses are not alive.
135
How could you determine if a bacterium is being killed by an antibiotic or its growth is simply being inhibited?
By removing the disc after incubation, if there is no growth it means the bacteria was killed but if growth is present then the bacteria was just inhibited.
136
Suppose that w/in the zone of inhibition for one drug, several small colonies were present after 24 hours incubation. You test these colonies and find that they are not contaminants. What is the presence of these small colonies?
?
137
In your own words explain; mutations, destruction or inactivation, and efflux.
Mutations-natural process that changes a DNA. Destruction-action or process of causing damage. Inactivation-process of no longer working Efflux-material flowing out.
138
In your own words, explain conjugation, transformation, and transduction.
Conjugation-act of joining together. Transformation-changing form or structure. Transduction-transfer of bacterial DNA from an infected bacterium to another.
139
In table 8-1, look up the phenol coefficient values of ethanol and Lysol. Which would be a better countertop disinfectant in a medical clinic? How do you know?
Ethanol would be a better countertop disinfectant in a medical clinic. It has a PC of 0.04.
140
A particular antimicrobial substance named "Microbe Masher" is listed as a "powerful antioxidant and microbe killer". You find the Microbe Masher kills Salmonella typhi at 10 minutes but not at 5 minutes at a concentration of 1 part "Masher" to 2 parts water. Phenol kills Salmonella typhi at 10 minutes but not at 5 minutes at a concentration of 1 part phenol to 100 parts water. What is the phenol coefficient of Microbe Masher? Which is the stronger disinfectant, phenol or Microbe Masher?
?
141
What happens when a protein is denatured?
They coagulate, uncoil and lose form.
142
What are the routine conditions for an autoclave?
15 pounds per square inch (psi) pressure, yielding a steam temperature of 121C for 15-30 minutes exposure.
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What are the limitations of an autoclave?
limited to materials that can only w/stand high temps and exposure to water.
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What is the most reliable way to monitor the effectiveness of an autoclave?
By the use of live spores of Geobacillus stearothermophilus in ampoules or on strips. After autoclave the ampoules/strips are incubated in nutrient broth. If sterilization is achieved the media will remain clear. Or use indicator tape that changes color after completion of a successful autoclave cycle.
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How would you sterilize a drug that is heat sensitive?
Using the method of filtration. Microorganisms are retained on the filter while the gas or liquid passes through the pores of the filter.
146
Cite some examples of how ultra-violet light is used as a biological control procedure?
Used in the final stages of waste-water treatment instead of other chemicals that could harm the environment. Can be used to sterilize utensils at your barber, hairstylist, or nail salon and in hospital operating rooms.
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What is the major limitation of ultra-violet light for disinfection?
Limited by poor penetration of liquids and solids. Limited at disinfecting air, surfaces, and transparent fluids.
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Is desiccation considered a static or cidal control procedure? Define both.
Desiccation or drying is an ancient method of control used in food preservation and works by depriving the microbe of water necessary for metabolic reactions such as glycolysis or protein synthesis. Considered a static method b/c many pathogens are resistant to the effects of drying, and only their growth is inhibited. Cidal-having power to kill Static-fixed
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Moist heat
More effective than dry heat. Moist heat denatures proteins by coagulation, while dry heat oxidizes proteins and dehydrates cells, slowly burning the microbe.
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What are the two most important variables in heat sterilization?
Time and temperature.
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Boiling
Relied on as only a disinfectant method.
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Autoclaving
Uses steam under pressure, achieving temperatures higher than that of boiling water. Routine autoclave conditions are 15 pounds per square inch (PSI) pressure, yielding steam temperature of 121C for 15-30 minutes exposure.
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Dry Heat
Includes incineration and hot air. Used for sterilizing inoculating loops and needles. Sterilization of biohazards waste, useful for glassware, instruments, and items such as talc that you would not want to get wet, and petroleum jelly, which the steam would not penetrate. Routine conditions would be 160-170C for two hours.
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Lyophilization
Modern method of drying that freezes the food or specimen and removes the water under a vacuum. Used to preserve microbial cultures for research or storage.
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Look at the slide w/ your eye, and then through the microscope. Does anything change? How does the microscope affect the orientation of the image?
Yes the slide is magnified making it appear larger. The ocular lenses magnify the image 10 times.
156
Explain how total magnification is calculated.
Multiplying the ocular lens by the magnification of the objective lens: 4x objective with 10x ocular = 40x magnification 10x objective with 10x ocular = 100x magnification 40x objective with 10x ocular = 400x magnification 100x objective lens with 10x ocular = 1000x magnification
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What effect does increased magnification have on the field of view? Field of view refers to the total amount of the object that you can see.
The field of view will decrease any time you magnify an object. Field of view representing the area of the specimen that will be visible though the ocular lenses when you are using different objective lenses.
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Looking at your resolving power calculations, explain the relationship between resolving power and resolution of your microscope. (pg. 91)
Resolution-ability to distinguish clearly between two adjacent objects, such as two bacterial cells side by side or very small, thin cell structures such as flagella. As you change to the next higher objective lens and increase the total magnification used to view your specimen, the resolution all increases. Clarity of the image is better.
159
Explain the difference between resolving power and the magnifying power of particular lens.
Resolving power-the resolution of each objective/ocular lens pair can be given a numerical value using the following formula: RP=wavelength/2xNA (objective lens). NA-numerical aperture refers to amount of light entering the objective lens from the specimen and this value increases your magnification.
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Explain why oil is necessary when using the 100x objective.
B/c light travels through the glass slide and comes in contact w/ the air it starts to bend and is directed away from the lens. This distorts the image. Oil can prevent refraction (bending) of light rays preventing them from coming into contact w/ the air, giving a clearer image.
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Procedures for cleaning microscope?
Always use lens paper to clean the lenses of the microscope. If lens is very dirty use only lens cleaner along w/ lens paper.
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Drawing/labeling observations?
Label colors of colonies, cell structures and indicate the colors of stained cells. Exaggerate the sizes of the cells that you are drawing so you can distinguish them. Label the drawing, point out to unique features, such as structures on or in specimens. Always show the total magnification used to view specimen.
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Rheostat knob
Adjust the light intensity
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Condenser
Group of lenses mounted below the stage of an optical microscope to concentrate light
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Iris Diaphragm
controls the amount of light entering the condenser.
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Ocular lens
Lens you look through to see the field of view.
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Parfocal
Once an object is in focus at low power it will remain in focus even when you move to a higher powered obj. lense.
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Fine adjustment knob
After focusing specimen at low power and you change the obj. lens you use the fine knob to bring the specimen into better detail.
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Parcentral
Once an object is centered in the field of view, it will remain close to or in the center when you move to higher powered obj.
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Objective lens
Nearest specimen, two systems of lenses that result in magnification.
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Ocular lens
Eyepiece, magnify's the image 10 times.
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Revolving nosepiece
Allow obj. lenses to rotate.
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Scanning obj. (4x)
scanning
174
10x obj.
low power
175
40x obj.
high power
176
100x obj.
oil immersion
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Field of View
total amount of the specimen that is visible, will decrease any time you magnify an object.
178
Identify two reasons why smears are heat-fixed.
1.) To kill bacteria, causing them to adhere to slide. 2.) Causes changes in the bacterial cells that enable them to take up stains more readily.
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Why are thick or dense smears less likely to produce a good smear preparation for microscopic evaluation?
If too many bacteria are present in the smear, clear differentiation of individual cells will be difficult. If to little bacteria are present the microscopist will have to spend a long time searching for a bacterial cell to view.
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Why is it essential that smears be air-dried rather then gently heated over a flame to speed up the drying process?
You will boil the organism if they aren't air-dried and cells will not stick to slide.
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Why should you be careful not to overheat the smear during the heat-fixing process?
Incinerated cells will not take up stain ad you will not see anything when you view the organism under the microscope.
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Bacteria cannot be seen w/ the naked eye or when viewed under the microscope, without staining. Why is fixing and staining important for the study of microorganisms?
It kills the bacteria so they can accept stains easily.
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As a microbiologist, when would you use the simple staining procedure?
When information is wanted about cell shape and size, as well as arrangement of the bacterial cells.
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Which part of the cell is the simple stain actually staining?
Gives color to the cell against a clear background b/c it is attracted to the overall negative charge of bacterial cells.
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Why are multiple stains required when performing a differential stain technique and only one stain is require for a simple stain?
Simple stain only requires one stain b/c of the overall negative charge of bacterial cells, the cationic stain binds w/ the cell, however, differences between cell components cannot be discerned. Good for looking at shape, size and arrangement Differential Staining Techniques-composed of two or more dyes and can differentiate between organisms' cellular components and therefore can allow the microscopist to differentiate between two different types of cells.
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Chromogens
Dyes that carry either a negative or positive charge.
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Cationic
Positively or basic charged stains, attracted to and stick to overall negative charge of a cell. Opposites attract.
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Anionic
Acidic stains, negatively charged, repelled by negative charge of cell.
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Negative Staining
Uses anionic stain when accurate determination of cell size/shape is needed.
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Common Cationic Stains?
Methylene blue, safranin, kinyoun's carbolfushsin, crystal violet.
191
Describe the structure and composition of a cell wall of Gram-positive Bacteria.
1.)Cell wall composed of peptidoglycan 2.)Multiple layers 3.)Negatively charged teichoic acids that extend throughout the cell walls. 4.)Lipteichoic acids-anchor peptidoglycan layer to cytoplasmic membrane.
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Describe the structure and composition of a cell wall of Gram-negative Bacteria.
1.) No teichoic acids 2.) Very thin cell wall 3.) Outer membrane linked to peptidoglycan 4.) lipopolysaccharide, sugars and lipid A 5.) More complex cell wall structure
193
What would happen if a student forgot to add the mordant during the gram stain?
The cells will not remain purple, the color will leach out.
194
Could bacteria that do not contain cell walls be stained using the Gram stain? Why or why not?
Cells without cell walls would not retain the dyes used in the gram stain procedure. The purpose of a gram stain is to determine the type of cell wall a certain bacteria has.
195
Could a counterstain other than safranin be used? Explain.
Yes, any dye that is cationic.
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Four basic steps in gram stain procedure? (pg. 115)
1.) Heat-fix smear is flooded w/ primary stain (crytal violet, a cationic stain turns purple) 2.) Add Mordant iodine, forms a water insoluble complex with CV-1 inside all cells, so all cells remain purple. 3.) Now wash cells w/ decolorizer (alcohol). Dissolves outer membrane of gram-negative and enhances removal of CV-1 through cell wall and Gram-negative become colorless. Gram positive's peptidoglycan layer collapses trapping the CV--1 and stay purple. 4.) Finally you counterstain bacteria w/ safranin, also a cationic stain (positive). Safranin stains the cells that are colorless, Gram-negative, and gives red/pink color, but doesn't affect gram-positive cells.
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What is the difference between a direct and indirect stain? These are also known as simple and negative stains.
Indirect (negative) stain-used to stain the background of a slide and not bacterial cells. Dark background provides contrast to make visualization of cells possible. "Stars in the night sky". No heat fix Direct (positive) stain-Direct staining, also referred to as simple staining, is when a single dye is used to stain a cell or cell component.
198
Which method, direct or indirect staining, do you think would present to you a better estimate of the true size of a microorganism? Explain reasoning.
Indirect staining, it does not require heat-fixing nor do the cells take up any stain. Cells do not shrink or become distorted w/ aggressive heating and permeation of stain. This is useful when accurate determination of cell size and shape is needed.
199
In terms of the charges of the stains and cell structures, when you perform a negative stain, why does the specimen appear light, while the background is stained black?
The india ink used, which is an anionic stain, is negatively charged, repelled by overall charge of bacterial cell, produces halo around cells.
200
India Ink
Uses in modified gin stain, india ink (anionic, - charge stain) produces an outline of capsule (cells shape) followed by crystal violet (counterstain, cationic, + charged stain) that will color the cell inside the thick capsule layer.
201
Crystal Violet
Uses in modified gin stain, will colorize the cell inside the thick capsule layer. The positively charged crystal violet will bind w/ the negatively charged cell inside the capsule. You'll see a gray background, a clear halo (capsule) around the cell and the violet-colored cell inside the capsule.
202
Why do you want to avoid mixing the cells w/ water?
Water will dissolve capsule
203
Why do you want to avoid heat fixing during the capsule staining?
Heat will destroy/distort capsule.
204
Why does the capsule make a bacterium virulent and more likely to cause a disease?
It can serve as a nutritional source when food is in short supply, prevents desiccation of the cell in dry environments. Protects bacterium by preventing phagocytosis by a white blood cell. Enhances adhesion, allowing it to stick to cells in the host.
205
How do capsules and slime layers differ?
Capsules are rigid and tightly bound to the bacterial cell. Slime layers (glycocalyx) can either be pliable and loosely bound to cell.
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Selective Pressures
Selection pressure can be regarded as a force that causes a particular organism to evolve in a certain direction.
