Microbiology Lab 3

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What is the purpose of a catalase test?

To identify organisms that produce the enzyme catalase.

What is the catalase test most commonly used for?

To differentiate members of the catalase-positive Micrococcaceae from the catalase-negative Streptococcaceae.

What does catalase do?

Converts hydrogen peroxide into water and gaseous oxygen.

How do you perform the catalase test?

Smear bacteria on slide and add hydrogen peroxide on top.
- If the bacteria is catalase-positive, then oxygen gas bubbles form immediately.
- If the bacteria is catalase-negative, then no bubbles appear.

What bacterias did we use in lab for catalase test?

- Staphylococcus aureus = positive
- Enterococcus (Streptococcus) = negative

Why does hydrogen peroxide bubble when it is placed into an open cut as a disinfectant?

Because catalase converts the hydrogen peroxide into water and gaseous oxygen.

What is the purpose of the oxidase test?

To identify bacteria containing the respiratory enzyme Cytochrome C Oxidase.

How is the oxidase test useful in identifying unknown species of Gram-negative rods?

It is useful in differentiating the oxidase-negative Enterobacteriaceae from the oxidase-positive Pseudomonadacae + Neisseria.

How do you perform the oxidase test?

- Smear bacteria on slide and add the reducing reagent.
- If the reducing agent becomes oxidized, the bacteria turns purple. Indicates a positive result.
- If the bacteria remains clear, the result is negative.

What bacterias did we use in lab for the oxidase test?

- E.coli = negative
- Pseudomonas = positive

What is the purpose of the nitrate reduction test?

To identify organisms that perform a one-step reduction from nitrate to nitrite using the enzyme nitrate reductase.

How is the nitrate reduction test useful in differentiating species of Gram negative rods?

It differentiates them from the Gram-negative rods that either do not reduce nitrate or reduce it beyond nitrite.

What does it tell you about the organism if it is positive for a nitrate reduction test?

It tells you that it contains the enzyme nitrate reductase and is able to perform a single-step reduction.

Possible end products of Nitrate Reduction

NO3 -> NO2 -> NO -> N2
-> NH4
-nitrate = NO3 = +5
-nitrite = NO2 = +3
-nitric oxide = NO = +2
-ammonia = NH4 = -3
- nitrogen gas = N2 = 0 = complete break down

Steps for Nitrate Reduction Test

1. Look in Durham Tube for nitrogen gas (a bubble at top) - if no gas, go to step 2
2. Look for nitrite. Add reagents A & B. If nitrite is present, a red ring forms at the top of the tube. If no red ring, the organism either could not convert nitrate into nitrite, or it converted into NO or NH4.
3. Add Zinc (catalyst) - it catalyzes the conversion of nitrate to nitrite. If red precipitate forms, NEGATIVE result b/c you made the conversion, not the bacteria!
4. If no red precipitate formed, the organism converted to either nitric oxide or ammonia. Smell it to see if it smells like ammonia.

What bacterias did we use in lab for the nitrate reduction test?

- Pseudomonas = N2 = 0
- Bacillus subtilis = NH4 = -3
- E.coli = NO2 = +3

What do biochemical tests look for?

Presence of enzymes.

What questions do biochemical tests answer?

1. What kind of information does this test give me?
2. How do I perform this test?
3. What does a positive and/or negative result look like?
4. What is the enzyme involved in each test?

What is the Phenol Broth Fermentation Test primarily used for?

To differentiate members of Enterobacteriacae and to distinguish them from other Gram-negative rods.

What does the fermentation tube have?

- Durham tube (to catch CO2)
- Color indicator (Phenol Red) (Turns red to yellow in acid pH)

What does the fermentation test test for?

- ability to ferment glucose
- if it can, the tube turns yellow and CO2 gas is trapped in the Durham Tube.
- look for growth on bottom of tube.

What bacterias did we use in lab for the Phenol Broth Fermentation Test?

- E.coli
- Proteus vulgaris
+ Tested for Glucose and Lactose

What was the color of the inoculated tube?

cloudy-yellow

What information is obtained from doing a Methyl Red Test?

Provides the ability to identify bacteria capable of producing stable stable acid end products by means of a mixed-acid fermentation of glucose.

Mixed Acid Fermentation

overcomes the phosphate buffer in the medium and lowers the pH.

How do you perform the Methyl Red Test?

1. Add methyl red indicator dye
- Red at pH 4.4 (acid); Yellow at pH 6.2 (alkaline)
- Red = positive
- Yellow = negative

What bacterias did we use in lab for the Methyl Red test?

- E.coli = positive
- Enerobacter aerogenes = negative

What information is obtained from doing a Voges-Proskauer Test?

Provides the ability to identify organisms capable of producing acetonin from the degradation of glucose during a 2,3-butanediol fermentation.

How do you perform the Voges-Proskauer Test?

1. Add reagents A & B and shake.
- If positive, red ring forms.
- If negative, no ring / copper ring forms.

What bacterias did we use in lab for the Voges-Proskauer Test?

- E.coli = negative
- Enerobacter aerogenes = positive

If positive for Methyl Red,

Negative for Voges-Proskauer.
- Exclusive test -

What information is obtained from doing the Starch Hydrolysis Test?

Which organisms are capable of reducing carbohydrate into simple sugars (amylase)

How do you perform the Starch Hydrolysis Test?

1. Inoculate on Starch Agar.
2. Add iodine to detect the presence or absence of starch.
- Iodine turns starch black = negative result
- Iodine turns amylase yellow = positive result
+ (This is testing for which bacteria is capable of hydrolyzing the starch down to amylase).

What bacterias did we use in lab for the Starch Hydrolysis Test?

- Bacillus subtillis = positive
- Pseudomonas arugenosa = negative
- E.coli = negative

What information is obtained from a Citrate Utilization Test?

It is used to determine the ability of an organism to use citrate as its sole source of carbon.

What are the results of the Citrate Utilization Test?

- Positive results turn the tube from green to blue.
- Negative results keep the tube green.

What was the color of the un-inoculated tube?

green

Why does the medium change in the Citrate Utilization Test?

- pH goes up (becomes alkaline) because of the production of ammonia and ammonium hydroxide.

What enzyme is used in the Citrate Utilization Test?

Citrate-permease

What bacterias did we use for the Citrate Utilization Test?

- E.coli = negative
- Enterobacter aerogenes = positive

What does a SIM tube test for?

1. Sulfur Reduction
2. Indole Production
3. Motility
+Use stabbing needle to inoculate+

What property does the Sulfur Reduction test determine?

It is used to differentiate members of Enterobacteriaceae, based on their ability to form H2S (hydrogen sulfite gas) using cysteine desulfurase and tiosulfate reductase.

What are the results of the Sulfur Reduction Test?

- If the organism is able to form H2S, the tube turns black (has black precipitate) = positive.
- If the organism is unable to form H2S, the tube is yellow = negative.

What bacterias did we use for the Sulfur Reduction Test?

- E.coli = negative
- Salmonella = positive
- Shigella = negative

What property does the Indole Test determine?

Identifies bacteria capable of producing indole using the enzyme tryptophanase.

How do you perform the Indole Test?

1. Stab the inoculating tube
2. Add Kovacs' Reagent (reacts with the indole)
3. Look for red ring
- If red ring present = positive
- If no red ring = negative

What bacterias did we use for the Indole Test?

- E.coli = positive
- Salmonella = negative
- Shigella = negative

What property does the Motility Test determine?

It is used to detect bacterial motility.
- feather-like pattern in SIM tube = positive.

What bacterias did we use in lab for the Motility Test?

- E.coli = positive
- Salmonella = unavailable b/c of sulfur reduction
- Shigella = positive

What property does the Gelatin Hydrolysis Test determine?

It is used to determine the ability of a microbe to produce gelitinase.

How does the Gelatin Hydrolysis Test work?

- The secreted gelatinase liquify the medium.
- Always do a control tube (should be negative!!!!)
- liquid = positive
- gel = negative

What bacterias did we use in lab for the Gelatin Hydrolysis Test?

- E.coli = (should be negative) (we got positive b/c incubator was off for a while)
- Proteus = positive
- Control = solid

What property does the Urease Test determine?

It is used to differentiate organisms based on their ability to hydrolyze urea with the enzyme urease.

How does the Urease Test work?

- Urea is hydrolyzes into ammonia in a positive test, turning the slant tube pink / fusia color.
- Negative result is a yellow / orange color.

What bacterias did we use in lab for the Urease Test?

- Proteus = positive
- Enterobacter = negative

What does IMViC stand for?

- Indole
- Methyl Red
- Voges-Proskauer
- Citrate

What is IMViC used for?

To distinguish between members of the Family Enterobacteriaceae and differentiate them from other Gram-negative rods.

Oxidase Test enzyme

Cytochrome C Oxidase

Catylase Test enzyme

Catylase

Nitrate Reduction Test enzyme

Nitrate Reductase

Citrate Test enzyme

Citrate-Permease

Sulfur Reduction Test enzyme

Cysteine Desulfurase
- and -
Thiosulfate Reductase

Indole Test enzyme

Tryptophanase

Gelatinase Test enzyme

Gelatinase

Urease Test enzyme

Urease

Which Tests are read-only's?

1. Fermentation Test
2. Citrate Test
3. Sulfur Reduction Test
4. Motility Test
5. Gelatin Hydrolysis Test
6. Urease Test

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