Lab Question Set 1 Flashcards
(133 cards)
1
Q
What are stains? What do they consist of?
A
- Stains are solutions that help make transparent cytoplasm and organelles visible
- Solutions consisting of a solvent and colored molecule
2
Q
What is a chromophore? An auxochrome? What kind of charge does the auxochrome have in basic stains?
A
- Chromophore - Portion of chromagen that gives color
- Auxochrome - Charged portion of chromagen
- Basic charges (+) are attracted to cell surface (-)
3
Q
Why are most bacteria most easily stained by basic stains (hint: think about the charges)?
A
- Most cells have a negative charge. Thus a basic charge (+) is attracted to the charge of bacteria
4
Q
Name 3 common basic stains.
A
Methylene Blue
Crystal Violet
Safranin
5
Q
List 3 reasons for heat-fixing bacterial smears.
A
- Kills bacteria
- Causes adhesion to slide
- Coagulates cytoplasm proteins increasing visibility
6
Q
Why is it usually necessary to stain bacterial cells in order to see them?
A
- Bacterial cells have transparent cytoplasms and by staining you can see cytoplasm as well as organelles
7
Q
In the medical laboratory, bacterial characteristics such as morphology and arrangement are usually determined by a _____________ stain.
A
Gram
8
Q
What is wrong with the heat-fixing technique illustrated on pg 186?
A
Allow the flame to completely pass through the flame and cool off on the other side. do not swipe it through the fire quickly.
9
Q
Why do we use a needle when making a smear from slant culture instead of using a loop?
A
Allows for more precise culture collection and avoids getting too much sample.
10
Q
How does the chromogen in a negative stain differ from the chromogen in the simple stain?
A
Negative stain has (-) charge that creates an outline to be able to see morphology, arrangement, and cell size
11
Q
The chromogen in a negative stain carries a ___________ charge. Is the negative stain, acidic or basic?
A
- Negative
- Negative stain is acidic
12
Q
Why do the bacterial cells remain unstained in a negative stain?
A
- The similar charges makes the stain repel from the cell
13
Q
Two stains often used in negative staining are ________________ and ____________________.
A
- Congo Red
- Nigrosin
14
Q
Negative stains are used to determine ____________________ and _______________________ in bacteria that are:
A
- Morphology
- Arrangement and cell size
- Fragile and heat sensitive
15
Q
A spirochete that is usually stained by a negative stain is ___________________________. This organism causes the disease __________________________. Another spirochete is ______________________________, which causes the disease _________________________.
A
- Treponema pallidum
- Syphilis (STI)
- Borrelia burgdorferi
- Lyme disease
16
Q
Why are negative stains used when determining the accurate size of a micro-organism is crucial?
A
Heat fixing can result in microorganisms shrinking. Negative staining does not
17
Q
In a good negative stain, what should the background of the slide look like?
A
Uniformly dark
18
Q
Simple stains stain the __________________ and not the _________________; negative stains stain the ______________________ and not the ________________________.
A
- Microorganism
- Background
- Background
- Microorganism
19
Q
What is wrong with the amount of acidic dye that the pg 192 recommends that you use? How much dye should you use?
A
A whole drop was used. A half drop can be used by touching the dropper to the slide.
20
Q
Differential stains allow a microbiologist to:
A
Differentiate gram (+) and gram (-) bacteria
21
Q
Which are used more frequently, differential stains or simple stains?
A
Differential Stains are used 95% of the time
22
Q
The most commonly used differential stain in bacteriology is the _______________ stain.
A
Gram
23
Q
Besides the gram stain, other differential stains test for the presence of _____________, _____________, _________________, or __________________ in bacteria.
A
- Acid-fastness
- Capsule
- Spores
- Flagella
24
Q
In the gram stain a _____________________ step occurs between the application of two ___________ stains.
A
- Decolorization
- Basic
25
The primary stain in the gram stain is _____________________. What do bacterial cells look like after application of this primary stain?
- Crystal violet
- Purple
26
Iodine acts as a ___________________ in the gram stain. What is the purpose of adding iodine?
- Mordant
- Binds negative charge to positive charge of crystal violet allowing more purple to stay on the cells
27
The most critical step in the gram stain is the ______________________ step.
Decolorization
28
The decolorizing solution we use in the gram stain is _______________________________.
Acetone Alcohol
29
In the last step in the gram stain, gram negative cells are colored by safranin, which is a ____________________________.
Red counterstain
30
The ability to resist or not resist decolorization in the gram stain is based on:
Composition of the cell wall in Gram positive and Gram negative cells
31
What do gram negative and gram positive cells look like before crystal violet is added in the gram stain?
They are transparent
32
What do gram negative and gram positive cells look like after the mordant is added in the gram stain?
- Gram positive cells will look purple
- Gram negative cells will be red
- Over exposure results in redish gram positive cells
- Underexposure results in purple gram negative cells
33
What do gram negative cells look like after the decolorization step in the gram stain? Why?
They will appear transleucent because both the crystal violet and iodine will have been washed off.
34
What do gram positive cells look like after the decolorization step in the gram stain? Why?
They will look purple because the crystal violet and iodine would have stained the cells and remained attached after the wash.
35
What will happen to gram negative cells if you don’t add enough alcohol in the gram stain?
They will maintain a red hue from the iodine
36
What will happen to gram positive cells if you add too much alcohol in the gram stain?
The gram positive cells will become more red in addition to purple giving poor visibility
37
Why is it best to use cultures that are 24 hours old or less for gram staining?
Bacillus and Staphylococcus can lose the crystal violet-iodine complex in more than 24 hours
38
Name 3 Gram negative bacterial genera and 2 Gram positive bacterial genera other than the genera used in this exp.
Gram Negative:
- Eschericha coli
- Citrobacter freundii
- Klebsiella pneumoniae
Gram Positive:
- Staphylococcus aureus
- Streptococcus pyogenes
39
What do the gram negative cells look like after successful completion of the gram stain? Gram positive cells?
- Gram positive cells will be purple
- Gram negative cells will be pink
40
Why are you asked to gram stain a mixture of gram positive and gram negative bacteria on the same slide?
To create clear contrast between gram positive and gram negative bacteria
41
Why do you need a counterstain in the gram stain?
1. Crystal violet attaches to both gram positive and negative cells
2. Mordant fixes crystal violet onto gram **positive** cells
3. Acetone alcohol Washes the crystal violet off gram **negative** cells
4. Saffranin **is then used to stain gram negative cells to provide visual contrast as the gram positive cells remain purple while the gram positive cells become pink**
42
Why do gram-negative bacteria stain differently than gram-positive bacteria in the gram stain? Be specific!
Gram Positive:
- Thick Peptidoglycan layer
- Crystal violet is retained by PG layer with the help of mordant even after acetone alcohol wash
Gram Negative:
- Thin Peptidoglycan layer
- Lipid A dense layer
- Crystal violet is washed out by acteone alcohol due to thinner PG and Lipid A layer
- Safranin is then used to stain the transparent gram negative cells
43
Capsules may be produced by certain bacteria and contribute to an increased ability to _______________ and adhere to surfaces.
Survive
(capsules can help bacteria stay hydrated)
44
Capsules are made of mucoid ____________________________ or ____________________________ that are secreted onto the ___________________ of bacterial cells.
- Polysaccharides
- Polypeptides
- Outer surface
45
Describe how capsules allow bacteria to survive for longer in their environment. Give at least 3 ways.
- Adherance factor (sticky)
- Antibiotic, antiseptic, disinfectant resistant
- Capsules allow for protection from dehydration and food source
- Anti-phagocytic properties
46
During the capsule staining procedure, congo red acts to stain the ______________________, while safranin stains the _______________________, leaving the capsule ________________________.
- Background
- Bacterial cell
- Transparent
47
The capsule stain employs both simple and negative staining procedures. Yet, the capsule does not absorb any dye, why might this be?
The capsule has a neutral charge
48
Why is hydrochloric acid applied during the capsule staining procedure? (2 reasons)
- Kills Bacteria
- Coagulates proteins
49
When working with potential pathogens, a capsule stain should be prepared with a beveled edge slide (with cut corners) to make the initial smear. How does the beveled edge help with safety?
It prevents bacterial sample from dripping over the edge of the slide
50
Some species of capsule-producing bacteria are environmental and non-pathogenic. However, there are some well-known diseases caused by bacteria that produce capsules. Name two diseases in this category and their causative agents.
Bacillus anthracis - Anthrax
Streptococcus pneumoniae - Pnuemonia
51
What is an endospore and what is it made of?
- Bacterial species that can differentiate into dormant cell when it encounters harsh conditions
- Endospores have a thick protective keratin coat that makes them very tough
52
Why are endospores resistant to heat and chemicals?
- Protective keratin layer
- Dehydrated state
- DNA protective proteins
53
Why must extreme measures be taken to stain endospores?
Because the keratin coat resists staining. Therefore spores must be steamed to soften and allow malachite green to enter
54
What is the difference between an endospore and a vegetative cell?
Endospores - metabolically dormant cell absent of ATP or any metabolic processes
Vegetative cells - metabolically active endospore cells
55
Name and describe 3 possible locations for endospores in the cell.
- Middle (central)
- Terminal (ends)
- Subterminal (between middle and end)
56
How old do the cultures of endospore-forming bacteria need to be before the bacteria start producing endospores?
Approximately 5 days
57
Endospores can be differentiated based on shape: they can be _______________ or ________________.
Spherical or elliptical (oval)
58
The 2 best-known genera of endospore-producing bacteria are:
Bacillus and Clostridium
59
List 4 diseased caused by endospore-forming bacteria and the genus and species of the pathogen that causes each disease.
*B.anthracis* - anthrax
*B.cereus* - food poisoning
*C.tetani* - tetanus
*C.botulinum* - botulism
*C.perfringens* - gas gangrene
60
If a basic stain like crystal violet is applied to a bacterial smear that contains endospores (without using steam), what will the endospores look like and why?
They would remain transparent as the thick protein (keratin) coat prevents staining
61
What will the vegetative cells look like (see question above) and why?
- Purple
- The protein coat would not be formed around a vegatative cell and therefore would stain
62
The process by which endospores return to the vegetative state is called _______________________.
germination
63
Under what conditions do bacteria produce endospores? What is the advantage of the endospores to the bacteria?
- Temperature, UV light, O2, pH, H2O, food stress
64
How long can endospores remain viable (capable of germinating into vegetative cells)?
Millions of years
65
Fermenters in this test ___________________ the medium more that do oxidizers. (____________ the pH)
- Acidify
- Lowering
66
Why does this medium have a high sugar-to-peptone ratio?
To reduce the possibility that alkaline products from peptone utilization will neutralize weak acids made from carb metabolization
67
The pH indicator in this medium is bromothymol blue, which is _____________ at pH 6.0 and ______________ at pH 7.1.
-Yellow
-Green
68
The lower-than-usual concentration of agar in this medium allows the student to determine ____________________ of the organism as well as its oxidative or fermentative abilities.
Motility
69
The fermentable carbohydrate that we have included in this medium is _______________________.
Glucose
70
After inoculation, one tube of O-F medium for each organism is sealed with a layer of mineral oil to promote ____________________________ and __________________________; the other tube for this organism does not get a layer of mineral oil, in order to promote ___________________________ and _______________________.
- Anaerobic growth
- Fermentation
- Aerobic growth
- Oxidation
71
If an organism grows in this medium, either with or without the mineral oil, and turns the medium blue, why did this change occur? What did the organism use as a food source? Is the appearance of a blue color in the medium a positive or negative test for the oxidation or fermentation of carbohydrate and why?
- The blue indicated a raised (alkaline) pH
- Peptones were used **not glucose**
- Negative test for both because oxidation of peptones occured not the breakdown of **carbohydrates**
72
What do you call an organism (oxygen requirements) that can ferment and oxidize the carbohydrate in this experiment (and which bacteria that we looked at fell in this category)? What do you call an organism that can oxidize the carbohydrate but not ferment it (and which bacteria that we looked at fell in this category)?
- Facultative anaerobe
- Escherichia coli
- Obligate aerobe
- Psuedomonas aeruginosa
73
Our OF medium contains glucose, but it could contain the carbohydrate __________________ or the carbohydrate ________________________.
- Lactose
- Sucrose
74
Acid-fast bacterial cell walls are produced by the genus __________________________. Two diseases caused by this genus include ________________________ and __________________________.
- Mycobacterium
- Lung tuberculosis
- Leprosy
75
Acid-fast cell walls are primarily made of ____________________ _________. Consequently, these cells are sticky and waxy.
- 60% mycolic acid
76
Describe how acid-fast bacteria can to survive for longer in their environment. Give at least 3 ways.
- Adherence factor
- Protection against phagocytic digestion
- Protects against dehydration
- Chemical protection (antibiotics and disinfectants)
77
Describe why carbolfuchsin dye must be heated and steamed during the acid-fast staining procedure.
- Carbolfuchsin is lipid soluble and penetrates the waxy cell wall when steamed as outer surface is softened
78
After the acid-fast staining procedure, acid-fast cells will have the color ____________________ while non-acid-fast cells will have the color _____________________.
- Pink
- Blue
79
During the last step of the acid-fast staining procedure, methylene blue is applied for 1 minute and then rinsed off. Explain why only some cells absorb the blue dye.
- Acid-Fast positive cells have mycolic acid layer that can resist methylene blue staining.
80
When working with acid-fast bacteria, an inoculating ________________ (rather than a needle) should be used to obtain and mix the bacteria into an emulsion during the smear preparation. Why is this important?
- Loop
- Becuase mycobacterium is sticky and would be hard to get on the stain with a needle
81
The non-pathogenic acid-fast genus and species of bacterium used in this experiment is called __________________ ____________________ and may cause an odor on the skin referred to as _________________ when it overgrows or is not washed by regular bathing.
- Mycobacterium smegmatis
- Smegma
82
_________________________ _________________________. Although it does not cause disease, it does cause a condition called _____________________ when it overgrows on human skin.
- Mycobacterium smegmatis
- Panniculitis
83
A “lactose fermenter” is an organism that splits the ____________________ lactose into _________________ and ______________________, and then ferments the ___________________________.
- Disaccharide
- Glucose
- Galactose
- Monosaccharides
84
The Methyl-Red (MR) test detects bacteria capable of performing a ______________________ fermentation.
Mixed acid
85
The Voges-Proskauer (VP) test identifies bacteria able to produce ________________________ as part of a ___________________________ fermentation.
- Acetoin
- 2,3-butanediol
86
Phenol red broth contains a fermentable ________________, ________________, and the pH indicator __________________________.
- Carbohydrate
- Peptone
- Phenol Red
87
Phenol red is ____________________ below pH 6.8 and _____________________ above pH 7.4.
- Yellow
- Pink/Magenta
88
____________________ of amino acids in the peptone in this broth produces ____________________, which __________________ the pH and turns the broth _________________ or _______________ (colors); the color of the broth may also remain the same. Do these results indicate a positive or negative test for the fermentation of the carbohydrate and why?
- Deamination
- Ammonia
- Increases
- Yellow or Pink
- Yellow is a positive test of fermentation as it indicates carb metabolism resulting in mixed acids being produced
- Pink is a negative test of fermentation as it indicates amino acid metabolism resulting in NH3 production
89
An inverted Durham tube (small test tube) is placed inside the culture tube to:
- Indicate gas production from fermentation
90
If the medium is examined after 18-24 hours of incubation, what does a yellow color indicate? Is this a positive or negative test for fermentation of the carbohydrate and why?
- Positive for carb fermentation
- Positive because phenol red indicator turns yellow in acid which is a byproduct of carb fermentation
91
To state that a particular organism is not capable of fermenting a particular carbohydrate (negative test), in addition to a color change, the medium must be _________________. If this last criterion is not met, why is it impossible to determine if that organism could ferment that carbohydrate?
- Cloudy
- There are two food sources. If it is not cloudy then neither food source was used
92
Why is it important to read these tubes after 24-48 hours after incubation? What color would the tube be after 48 hours? Why?
Because at 24 hours both the Methyl Red and Voges-Proskauerare acidic. After 48 hours, acetoin will be produced in the VP. Both the MR and VP will be pink/red if pH decreases and acetoin is present in each respective test tube.
93
Glucose is included in MR-VP medium as a __________________________; the purpose of the phosphate buffer is:
- Substrate (fermentable carb)
- Resist pH change
94
The MR test is designed to detect organisms capable of _______________________ the buffer and ________________________________________.
- Overcoming
- Lowering the pH
95
Bacteria that are MR+ carry out what is called a ______________________________________. The acids produced by these organisms tend to be ________________, whereas acids produced by other organisms tend to be:
- Mixed acid fermentation
- Stable
- Unstable and become neutral or alkaline
96
The pH indicator methyl red is _______________ at pH 4.4 and _______________ at pH 6.2 (note that this is exactly the opposite of the color range of the pH indicator __________________). Between pH 4.4 & pH 6.2, methyl red is various shades of _____________________.
- Red
- Yellow
- Phenol red
- Orange
97
____________ color is considered the only true indication of a + result in the MR test; orange broth is considered _________________ or_________________. _______________________ is negative.
- Red
- Negative
- Inconclusive
- Yellow
98
The VP test is designed to identify organisms that ferment glucose but quickly convert their acid products to ___________________ and ___________________________.
- Acetoin
- 2,3-butanediol
99
A positive VP test is ________________ (color); no color change is _______________________.
- Red
- Negative
100
A copper color in the VP test is the result of interaction between the reagents and should not be confused with the true __________________ color of a + result.
- Red
101
If an organism that cannot ferment glucose, grows in this broth, what did it use for a food source?
- Peptone
102
Why are organisms that are MR+ usually VP-, and VP+ organisms usually MR-?
- Because the organisms fermenting glucose either yields stable acids or acetoin which is more alkaline. Rarely does an organism produce both.
103
The MR and VP tests are components of a battery of tests known by the initials ____________________. Indicate what each of these letters stands for:
- IMViC
104
All fermentation pathways start with the compound ____________________, produced by _______________________. (process)
- Pyruvate
- Glycolysis
105
The only carbon-containing compound in citrate medium is ______________________. Why do organisms need a carbon source?
- Citrate
- To make all macro molecules
106
The only nitrogen source in citrate medium is ________________________. Why do organisms need a nitrogen source (to make which 2 macromolecules)?
- Ammonium dihydrogen phosphate
- Amino acids and nucleotides (DNA, RNA)
107
In a medium where citrate is the only available carbon source, bacteria that possess the enzyme ________________________________ can transport the citrate molecules into the cell and metabolize them.
- Citrate Permease
108
The pH indicator in citrate medium, bromothymol blue, is _________________ at pH 6.9 and ________________ at pH 7.6.
- Green
- Blue
109
Bacteria that survive in this medium and utilize the citrate create an _____________________ (increase or decrease?) in the pH of the medium.
- Increase
110
Describe a positive result in this medium. Why does the pH indicator in the medium change color in a positive test? Indicate the name of the product and the relative pH (acidic or basic) that caused the color change.
- Positive test shows utilization of citrate through breakdown of Ammonium dihydrogen phosphate which causes increase in pH leading to **blue color change**
111
Describe a negative result in this medium. Why is there no growth in a negative result?
- Negative is green **with no growth**. if it is green with growth it is still positive as there likely has not been enough alkaline products produced yet
- There will be no growth as citrate could not be utilized and no alkaline products were produced
112
If there is growth on citrate medium after incubation but no color change, this is considered a ________________________ result.
Positive
113
Citrate utilization is one part of a test series referred to as IMViC; these letters stand for the 4 tests:
I: Indole
M: Methyl Red
Vi: Voges-Prskauer
C: Citrate
114
The presence of ammonium hydroxide in the medium is INDIRECT evidence that the bacterium has broken down __________.
- Citrate
- Nitrogen is necessary for growth as is carbon so if basic products are produced it indicates the use of citrate as well.
115
What is a polysaccharide?
A polysaccharide is a type of macromolecule made up of many sugar units.
116
Why will starch not pass through the bacterial cell (plasma) membrane?
Starch is too large to pass through the bacterial cell membrane.
117
Bacterial enzymes that can hydrolyze starch are __________ enzymes, namely __________ and __________.
Bacterial enzymes that can hydrolyze starch are amylolytic enzymes, namely alpha-amylase and beta-amylase.
118
The overall reaction for the hydrolysis of starch involves breaking the polysaccharide down to __________.
The overall reaction breaks starch down to glucose and maltose.
119
Why is it not possible to see either starch or its sugar subunits in starch agar?
They are either too large or too soluble to be visible in the agar.
120
What is the purpose of adding gram’s iodine to starch agar plates after incubation?
The purpose is to visualize starch hydrolysis by staining the agar.
121
Describe a positive result in this experiment.
A positive result shows a clear zone around the colonies, indicating starch hydrolysis.
122
Describe a negative result in this experiment.
A negative result shows no clear zone, indicating no starch hydrolysis.
123
After adding iodine, what would an uninoculated plate of starch agar look like? Why?
It would appear blue-black due to the presence of iodine staining the starch.
124
Starch hydrolysis is one of the tests that aids in the differentiation of species in the genus Clostridium, members of which cause the diseases __________ and __________, and species in the genus Bacillus, which causes __________.
Members of Clostridium cause botulism and tetanus, while Bacillus causes anthrax.
125
What do the 2 bacterial genera mentioned have in common?
Both genera are anaerobic and can form spores.
126
What conclusion can you draw from the fact that after iodine has been added, the clear area on starch agar around the colonies of the organism that is + for starch hydrolysis is only found a short distance from the colonies? If the organism is + for starch hydrolysis, why isnt the whole plate clear after iodine has been added?
- The bacteria release amylase into their environment and have not been given enough time to grow out. Given enough time the plate could become clear when iodine is added.
127
ONPG is a synthetic artificial chemical that contains _____________ sugar on one half of its disaccharide-like structure. The disaccharide called ______________ most resembles ONPG.
- Beta-Galactose
- Lactose
128
ONPG is digested by bacteria that produce the enzyme _____________________________.
Beta-Galactosidase
129
ONPG is often used in place of the equivalent Phenol Red Lactose test because results can be read in as little as _________hours.
Two
130
ONPG is the substrate in this test and it starts out the color ______________. Following digestion by the bacteria, the color ______________ will indicate a positive result when ________________ is produced.
- White
- Yellow
- o-Nitrophenol
131
Explain how ONPG yields faster results than the Phenol Red Lactose test. (Hint: Lactose permease should be mentioned in your answer)
- It faster because Phenol Red takes 24 hours to make lactose permease to get into the cell
132
Why is ONPG considered more accurate than the equivalent Phenol Red Lactose test?
- It is permease independent because bacteria can mutate and no longer have functional lactose permease gene
133
The ONPG is a rapid test that only requires water, an ONPG tablet, and a heavy inoculum of bacteria. Why is it okay to handle the ONPG tablet with your hands (questionable aseptic technique) before placing the tablet in the
test tube?
- Due to the test being rapid, the possible bacteria on your hand will be negligible and outnumbered compared to millions form inoculum
