lab practical 2

What is the purpose of the phenol red in the carbohydrate fermentation broth?

It is an acid indicator

If the acid is produced it will turn yellow

What is the purpose of the Durham tube and the carbohydrate fermentation broth?

It is a gas indicator

If gas is produced it will leave a gas bubble in the Durham tube

What are the three possible in products of fermentation?

Acids alcohol gasses

What is the main product from the catalyst test causing the bubbling

Oxygen

What bacterial genera are differentiated using the catalase test?

Enterococcus

Staphylococcus

Great for gram +

Would an obligate anaerobe be positive for catalase? how do you know?

Not positive

they must live in an environment without O2

they lack catalase

they don’t need oxygen

The oxidase test Is used to differentiate between which groups of bacteria?

Obligate Aerobes and facultative anaerobes

Why should a metal loop not be used to transfer bacteria in the oxidase test?

The metal will transfer the electrons causing false positives therefore you need to use wooden sticks metal is a great conductor

 

What in product reacts with nitrate a and nitrate b reagents?

Nitrite

What is the function of zinc in the nitrate test?

Reduces NO3 to NO2

If nitrate is still there it turns red

1. An unknown bacterium was inoculated into the sucrose fermentation tube was sucrose fermented

2. What are two of the three end products that you can detect with this test

3. What is the function of the Durham tube in the tube

1. Yes-yellow

   no-red

   Gas bubble-gas production

   No gas bubble- no gas production

2. Acid, alcohol, gas

3. Gas detection

   Catches gas

1. Methyl red has been added to this tube. Is this a positive or negative test

2. Is this bacterium a butanediol fermenter or a mixed acid fermenter?

1. Red-positive

   Yellow-negative

2. Mixed acid- positive for methyl red

   Butanediol- positive for VP test

This is the sim tube

1. Is this bacterium positive or negative for the sulfur test

2. is this bacterium positive or negative for the indole test

3. What is the substrate in this reaction that this bacterium is using for food?

4. Is this bacterium motile?

1. black-positive

   clear-negative

2. red on top-positive

   yellow or amber on top-negative

3. Tryptophan

4. Cloudy-positive

   Not cloudy-negative-grows only in the line

1. Perform this catalase test is this bacterium positive or negative for this test

2. What oxygen radical does this test destroy

1. 1.       Place a drop of catalase reagent onto glass slide

   2.       Use inoculating loop, transfer small amount of culture to drop on slide

   Bubbles-positive

   No bubbles-negative

2. Hydrogen peroxide

1. Can this bacterium utilize citrate as its sole carbon source?

2. Name one of the two enzymes necessary for this reaction to occur

1. Blue-yes/positive

   Green-noegative

2. Citrase

   Citrate permease

This is the gelatin test it has been refrigerated at four degrees Celsius for 20 minutes

1.  Is this test positive or negative

2. If a bacterium is positive for the gelatin test what would the tube look like

3. Why is this test important in the health care field

1. Liquid-positive

   Solid-negative

2. At 4 degrees Celsius: liquid

   At 37 degrees Celsius: liquid

3. Gelatin can be used as a food source allowing bacteria to eat or degrade connective tissue and spread

1. What does IMViC stand for

2. What group of bacteria would these tests help to identify

3. This is the urease test does this bacterium contain urease

4. Name one reason containing urease could be beneficial to the bacterium

1. Indole

   Methyl red

   Vogues-Proskauer

   Citrate

2. Enterobacteria

3. Hot pink-positive

   Yellow-negative

4. pH of environment

   Can breakdown urea as a nitrogen or food source

1. This is the oxidase test Is this bacterium positive or negative for this test

2. Why is a wooden stick required to transfer the bacteria to the filter paper

3. Perform the oxidase test

4. What two groups of bacteria can be differentiated using this test

1. Purple/pink- positive

   Remain same color-negative

2. Metal loop could oxidize creating a false positive

3. 1.       Add a drop of the oxidase reagent to the oxidase paper

   2.       Using sterile wooden stick transfer a large amount of a culture onto the oxidase test paper you need to be able to see where you place the cells on the filter return the wood tool a used stick test tube

4. Obligate aerobes and facultative anaerobes

1. This is the DNase test is this bacterium positive or negative for this test

2. To what general category of enzyme does DNase belong

3. Why is this test important in the health field

1. Clearing-positive

   No clearing-negative

2. Exoenzyme

3. DNase can allow bacteria to degrade host DNA to increase pathogenicity and spread

 Two unknown bacteria were inoculated into a nitrate tube. nitrate A and B were added to tube #1 and nitrate A and B and zinc were added to tube #2

1. Does the bacterium and tube number one possess nitrate reductase

2. Does the bacterium and tube number two possess nitrate reductase

3. What does the bacterium use nitrate for if it contains nitrate reductase

1. Red-yes/ positive

   No change- ? need to do step 2

2. Red-negative

   no change/clear-positive

3. Final electron acceptor

1. In the SIM test the presence of a black precipitate could indicate two different beneficial reactions in the tube name the two reasons a bacterium might create a positive reaction

2. What end product combines with iron to form the black precipitate

1. 1.       Cysteine for food source

   2.       thiosulfate as final electron acceptor

2. H2S

1.  This is the starch test is this bacterium able to hydrolyze starch

2. What reagent is added in order to identify whether this is a positive or negative test

3. What product of this reaction can be transported across the cell membrane

1. Clearing-yes/positive

   No clearing-no/negative

2. Iodine

3. Glucose and maltose

This is the Vogues-Proskauer test

1. Is this test positive or negative

2. What does this test tell you about the bacterium

3. What is one error that can be made in this test to give you incorrect results

1. Positive- red

   Negative-yellow

2. It is not a butanediol fermenter-negative

   It is a butanediol fermenter-positive

3. Not dividing into 1/3 (VP) and 2/3 (MR)

You have allowed bacteria from the lake to incubate overnight in Luria broth

1. How would you isolate the bacteria in the sample so that you get individual bacterial species

2. What is the first thing you will do with the culture after you’ve grown it up in order to identify it

3. Where would you find test results that would help you to identify your unknown bacterium besides your lab manual

1. Streak plate

2. gram stain

3. bergeys manual

1. Why is this plate white

2. Is this bacterium able to produce protease

3. How can a bacterium grow on a milk plate if they do not produce proteases

1. Contains casein a white milk protein

2. Zone of clearing-yes/positive

   No zone of clearing-no/negative

3. Utilize other nutrients to grow and survive that are on the plate

How much was in the original sample

black throughout and red on top

sulfide, indole, motility?

+,+,+

black in a line and yellow on top

sulfide, indole, motility?

+.-,-

cloudy throughout and yellow in top

sulfide, indole, motility

-,-,+

A student inoculates a SIM tube for a hydrogen sulfide production test. After the incubation the student notices a black precipitate in the tube. The student concludes that only thiosulfate was reduced for the test. Student correct? Why not?

no, it could be positive for cystein or thiosulfate because both have a black precipitate.

what does the methyl red reagent detect?

Mixed acid fermenters

pH change

A student inoculates and incubates a vogues Proskauer test. the following day the student removes the tube from the incubator and adds Barritts reagent A and Barritt's reagent B. the Organism should be positive for VP but the test is negative. What is wrong with the test

didnt divide the media- not correct ration

what enzymes do bacteria require to use citrate as a carbon source

1. citrate permease

2. citrase

what does a positive citrate test look like

bright blue color

what are the benefits of the urease enzyme to the bacteria

can break down urea as a nitrogen source (food source)

also raise pH of environment

in what clinical infection would a urease test be used for diagnosis

UTIs

what is the importance of exoenzymes to bacteria

Enzymes released to allow for extracellular digestion.

Pull smaller across cell membrane.

gives food Source that is extra large.

Large particle into smaller and across cell membrane

a-amylase is an enzyme that breaks down starch. what products of this reaction can be transported across the cell membrane

1. maltase

2. glucose

what reagent must be added to the starch plate to identify if a-amylase was produced

iodine- zone of clearing

what enzyme must be produced to degrade casein

protease enzyme

why are some bacteria able to grow on a milk plate even though they are not able to hydrolyze casein

they can grow due to other stuff in the milk

used as food but might not have protease enzyme

so it can hydrolyze casein

what are two reasons that the production of gelatinase is beneficial to certain bacteria

virulence factor- can move and have food source everywhere

food source- breaks down connective tissue as food

1. food

2. movement

how is gelatin test performed

1. inoculate- half way in and swirl

2. incubate 48 hrs at 37 degrees Fahrenheit- melts

3. refrigerate 20 min at least- allow to see if hydrolyzed

4. read results

what does a positive gelatin test look like

wont resolidify- hydrolyzed- liquid after refrigeration

what does a positive DNAse test look like

showing zone of clearing around bacteria

why is DNAse a virulence factor

every cell has DNA if it can break down host DNA it can easily kill host