Lab Quiz 2 Flashcards by Rene Galindo

What is the purpose of conducting an endospore stain?

3-10 Endospore Stain

Allows visualization of endospores
Allows us to differentiate between spore forming cells and non-spore forming cells

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What two bacteria produce endospores? Are these gram +/-?

3-10 Endospore Stain

Bacillus and Claustridium
Gram positive

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Outline the steps in the cycle of a spore encountering unfavorable then favorable conditions.

3-10 Endospore Stain

Mother cell is active
Harsh conditions promote sporulation
Endospore forms within mother cell and is protected (can survive millions of years
Spore encounters favorable germination conditions leading it to become a mother cell

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Capsules may be produced by certain bacteria and contribute to an increased ability to _______________ and adhere to surfaces.

3-9 Capsule Stain

Survive
(capsules can help bacteria stay hydrated)

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Capsules are made of mucoid ____________________________ or ____________________________ that are secreted onto the ___________________ of bacterial cells.

3-9 Capsule Stain

Polysaccharides
Polypeptides
Outer surface

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Describe how capsules allow bacteria to survive for longer in their environment. Give at least 3 ways.

3-9 Capsule Stain

Adherance factor (sticky)
Antibiotic, antiseptic, disinfectant resistant
Capsules allow for protection from dehydration
Anti-phagocytic properties

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During the capsule staining procedure, congo red acts to stain the ______________________, while safranin stains the _______________________, leaving the capsule ________________________.

3-9 Capsule Stain

Background
Bacterial cell
Transparent

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The capsule stain employs both simple and negative staining procedures. Yet, the capsule does not absorb any dye, why might this be?

3-9 Capsule Stain

The capsule has a neutral charge

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Why is hydrochloric acid applied during the capsule staining procedure? (2 reasons)

3-9 Capsule Stain

Kills Bacteria
Coagulates proteins

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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?

3-9 Capsule Stain

It prevents bacterial sample from dripping over the edge of the slide

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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.

3-9 Capsule Stain

Bacillus anthracis - Anthrax
Streptococcus pneumoniae - Pnuemonia

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What is an endospore and what is it made of?

3-10 Endospore Stain

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

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Why are endospores resistant to heat and chemicals?

3-10 Endospore Stain

Protective keratin layer
Dehydrated state
DNA protective proteins

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Why must extreme measures be taken to stain endospores?

3-10 Endospore Stain

Because the keratin coat resists staining. Therefore spores must be steamed to soften and allow malachite green to enter

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What is the difference between an endospore and a vegetative cell?

3-10 Endospore Stain

Endospores - metabolically dormant cell absent of ATP or any metabolic processes
Vegetative cells - metabolically active endospore cells

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Name and describe 3 possible locations for endospores in the cell.

3-10 Endospore Stain

Middle (central)
Terminal (ends)
Subterminal (between middle and end)

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How old do the cultures of endospore-forming bacteria need to be before the bacteria start producing endospores?

3-10 Endospore Stain

Approximately 5 days

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Endospores can be differentiated based on shape: they can be _______________ or ________________.

3-10 Endospore Stain

Spherical or elliptical (oval)

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The 2 best-known genera of endospore-producing bacteria are:

3-10 Endospore Stain

Bacillus and Clostridium

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List 4 diseased caused by endospore-forming bacteria and the genus and species of the pathogen that causes each disease.

3-10 Endospore Stain

B.anthracis - anthrax
B.cereus - food poisoning
C.tetani - tetanus
C.botulinum - botulism
C.perfringens - gas gangrene

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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?

3-10 Endospore Stain

They would remain transparent as the thick protein (keratin) coat prevents staining

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What will the vegetative cells look like (see question above) and why?

3-10 Endospore Stain

Purple
The protein coat would not be formed around a vegatative cell and therefore would stain

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The process by which endospores return to the vegetative state is called _______________________.

3-10 Endospore Stain

germination

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Under what conditions do bacteria produce endospores? What is the advantage of the endospores to the bacteria?

3-10 Endospore Stain

Temperature, UV light, O2, pH, H2O, food stress

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How long can endospores remain viable (capable of germinating into vegetative cells)? ## Footnote 3-10 Endospore Stain

Millions of years

Fermenters in this test ___________________ the medium more that do oxidizers. (____________ the pH) ## Footnote 5-2 OF Test

- Acidify - Lowering

Why does this medium have a high sugar-to-peptone ratio? ## Footnote 5-2 OF Test

To reduce the possibility that alkaline products from peptone utilization will neutralize weak acids made from carb metabolization

The pH indicator in this medium is bromothymol blue, which is _____________ at pH 6.0 and ______________ at pH 7.1. ## Footnote 5-2 OF Test

-Yellow -Green

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. ## Footnote 5-2 OF Test

Motility

The fermentable carbohydrate that we have included in this medium is _______________________. ## Footnote 5-2 OF Test

Glucose

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 _______________________. ## Footnote 5-2 OF Test

- Anaerobic growth - Fermentation - Aerobic growth - Oxidation

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? ## Footnote 5-2 OF Test

- 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**

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)? ## Footnote 5-2 OF Test

- Facultative anaerobe - Escherichia coli - Obligate aerobe - Psuedomonas aeruginosa

What do you call an organism that cannot ferment or oxidize carbohydrates? ## Footnote 5-2 OF Test

- Non-saccharolytic - Break down peptones (blue)

Our OF medium contains glucose, but it could contain the carbohydrate __________________ or the carbohydrate ________________________. ## Footnote 5-2 OF Test

- Lactose - Sucrose

A “lactose fermenter” is an organism that splits the ____________________ lactose into _________________ and ______________________, and then ferments the ___________________________. ## Footnote 5-3 Phenol Red Broth

- Disaccharide - Glucose - Galactose - Monosaccharides

The Methyl-Red (MR) test detects bacteria capable of performing a ______________________ fermentation. ## Footnote 5-3 Phenol Red Broth

Mixed acid

The Voges-Proskauer (VP) test identifies bacteria able to produce ________________________ as part of a ___________________________ fermentation. ## Footnote 5-3 Phenol Red Broth

- Acetoin - 2,3-butanediol

Phenol red broth contains a fermentable ________________, ________________, and the pH indicator __________________________. ## Footnote 5-3 Phenol Red Broth

- Carbohydrate - Peptone - Phenol Red

Phenol red is ____________________ below pH 6.8 and _____________________ above pH 7.4. ## Footnote 5-3 Phenol Red Broth

- Yellow - Pink/Magenta

____________________ 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? ## Footnote 5-3 Phenol Red Broth

- 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

An inverted Durham tube (small test tube) is placed inside the culture tube to: ## Footnote 5-3 Phenol Red Broth

- Indicate gas production from fermentation

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? ## Footnote 5-3 Phenol Red Broth

- Positive for carb fermentation - Positive because phenol red indicator turns yellow in acid which is a byproduct of carb fermentation

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? ## Footnote 5-3 Phenol Red Broth

- Cloudy - There are two food sources. If it is not cloudy then neither food source was used

Why is it important to read these tubes after 24-48 hours after incubation? What color would the tube be after 48 hours? Why? ## Footnote 5-3 Phenol Red Broth

Because at 48 hours digestion of peptones may begin to occur as carb food source decreases. This will lead to a pH rebound as the sample goes from acidic to basic.

Glucose is included in MR-VP medium as a __________________________; the purpose of the phosphate buffer is: ## Footnote 5-4 Methyl Red and Voges-Proskauer Test

- Substrate (fermentable carb) - Resist pH change

The MR test is designed to detect organisms capable of _______________________ the buffer and ________________________________________. ## Footnote 5-4 Methyl Red and Voges-Proskauer Test

- Overcoming - Lowering the pH

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: ## Footnote 5-4 Methyl Red and Voges-Proskauer Test

- Mixed acid fermentation - Stable - Unstable and become neutral or alkaline

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 _____________________. ## Footnote 5-4 Methyl Red and Voges-Proskauer Test

- Red - Yellow - Phenol red - Orange

____________ color is considered the only true indication of a + result in the MR test; orange broth is considered _________________ or_________________. _______________________ is negative. ## Footnote 5-4 Methyl Red and Voges-Proskauer Test

- Red - Negative - Inconclusive - Yellow

The VP test is designed to identify organisms that ferment glucose but quickly convert their acid products to ___________________ and ___________________________. ## Footnote 5-4 Methyl Red and Voges-Proskauer Test

- Acetoin - 2,3-butanediol

A positive VP test is ________________ (color); no color change is _______________________. ## Footnote 5-4 Methyl Red and Voges-Proskauer Test

- Red - Negative

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. ## Footnote 5-4 Methyl Red and Voges-Proskauer Test

- Red

If an organism that cannot ferment glucose, grows in this broth, what did it use for a food source? ## Footnote 5-4 Methyl Red and Voges-Proskauer Test

- Peptone

Why are organisms that are MR+ usually VP-, and VP+ organisms usually MR-? ## Footnote 5-4 Methyl Red and Voges-Proskauer Test

- Because the organisms fermenting glucose either yields stable acids or acetoin which raises the pH. Rarely does an organism produce both.

The MR and VP tests are components of a battery of tests known by the initials ____________________. Indicate what each of these letters stands for: ## Footnote 5-4 Methyl Red and Voges-Proskauer Test

- IMViC

All fermentation pathways start with the compound ____________________, produced by _______________________. (process) ## Footnote 5-4 Methyl Red and Voges-Proskauer Test

- Pyruvate - Glycolysis

What is the purpose of the Phenol Red Broth experiment? ## Footnote 5-3 Phenol Red Broth

To determine which bacteria can metabolize a specific carbohydrate. - In exp. we used a mono and disacch to see which organism could break them down.

What do the possible colors and turbitity mean in phenol red broth test indicate? ## Footnote 5-3 Phenol Red Broth

Yellow - Acid was produced (+) Red/orange - neutral Pink - Alkaline (-) In order for a result to be conclusive it must be cloudy to indicate bacterial growth, otherwise it is retest. Even if yellow and clear **must be cloudy**

What is the purpose of the OF test? ## Footnote 5-2 OF Test

- Test if bacteria oxidize and or ferment glucose

What is the pH indicator in O/F test? What do the colors mean? ## Footnote 5-2 OF Test

Bromothymol blue - Yellow is acidic - Green is neutral - Blue is alkaline

What does aerobic processing of glucose result in? Anaerobic? ## Footnote 5-2 OF Test

- Acids - Acids, alcohol, +/- gases

What do these results tell us ## Footnote 5-2 OF Test

- Strick oxidizer of glucose - Oxidizer and fermenter of glucose (facultative prefers O2 but can function anaerobically) - Non-oxidizer and non-fermender of glucose (non-saccharolytic doesn use sugars but peptones instead)

What is the purpose of an indole test? What is a positive test? Negative? ## Footnote Indole

- To test if bacteria can produce indole from tryptophan - Pink Ring after Kovac's reagent indicates indole - Yellow ring after Kovac's indicates no indole