MicroBiology Lab Practical 2 Blinn McNeil

1. Identify various media and what is tests for if given a +/- reaction and an uninoculated control.

- Use pictures of reactions in lab manual, experience with practice inoculations and any demos shown in lab.

- Remember if a medium is plate media, broth media, slant media, or deep media.

2. List substrates, products, or enzymes detected or used in differential or selective media.

- The substrate hydrolyzed (broken down) in phenol red glucose broth is glucose.

- The enzymes detected in Simmon's citrate agar are citrate permease and citrase.

3. Identify if a given medium is selective or differential and list the conditions that make it so.

- MacConkey agar is selective for Gram Negative organisms due to the presence of bile salts and crystal violet and differential based on if those Gram (-) organisms can ferment lactose.

- Mannitol salt agar is selective for staphylococci and differential based on if those staphylococci can ferment mannitol.

4. Know the color changes for both positive and negative reactions and be able to interpret those reactions.

- What does a yellow phenol red glucose tube indicate? What does a pink tube indicate? Orange tube?

- Which of these color changes indicates a (+) reaction for fermentation? Which indicates a (-) reaction for fermentation?

5. List any indicators used in media and what their color changes imply?

- The pH indicator in MacConkey agar is neutral red. Red growth on this agar means that the pH has decreased and the test is (+) for lactose fermentation.

6. List any reagents added to media ( know reagent names, not just "reagent A" or "reagent B".

- The reagent (and substrate) for the catalase test is hydrogen peroxide.

- The reagents used in the nitrate test (A) sulfanilic acid and (B) napthylamine for step 1 and (C) zinc for step 2.

7. List the steps of a test if you were asked questions about the procedure or asked to actually perform the test.

- Only applies to "rapid" tests like oxidase, catalase, or coagulase.

8. Critical Thinking: be able to apply what you know about one biochemical test to infer results about another test.

- Based on the results of your MacConkey agar which showed red colonies, what would the result of your phenol red lactose broth be?

9. Know the key biochemical or morphological characteristics of the different bacterial families and organisms discussed in each lab.

Examples:

- Lancefield classification for streptococci

- Staphylococci

- Gram Negative enteric coliforms

Lab 17:

Selective Media:

- these types of media contain compounds that inhibit the growth of some organisms, while allowing the growth of others.

To grow only one particular group of microorganisms that is present in a mixed population of organisms.

Differential Media:

- contain substances that are utilized differently by microorganisms and allow the differentiation between organisms based on their particular metabolic capabilities.

Used to differentiate between organisms growing in a culture.

Selective and Differential Media:

- In some circumstances a medium can be both selective and differential.

- Selective and differential media select for specific organisms, and then differentiate between the organisms capable of growing under those selective conditions.

Tryptic soy agar (TSA):

- a complex medium which can be used for the isolation of most microbes.

- Excellent growth medium that contains digests of casein (milk protein) and soybean meal as sources of carbon and energy, glucose as energy, and various salts.

- allows for the growth of variety of microorganisms from both clinical and nonclinical specimens.

Lab 18:

Phenol Red Broth

Phenol Red Broths: (Lab 18)

Differentiates organisms based on ability to ferment carbohydrates.

Media

- Glucose - Phenol Red Broth

- Lactose - Phenol Red Broth

- Sucrose - Phenol Red Broth

- Mannitol - Phenol Red Broth

Durham Tube:

- small glass tube inverted in the bottom of the tube to determine if some bacteria produce gas (fermentation).

Reversion:

- If the organism expresses the enzymes for catabolizing peptones (found in proteins), it can continue to produce ATP and will liberate ammonia (NH3), NH3 causes a rise in the pH and the phenol red indicator turns bright pink.

Deamination:

- Some non-fermenting organisms are capable of deamination, possessing enzymes that can hydrolyze the peptone amino acids in the media, releasing NH3.

Color Change:

- "A" Acid - Yellow color

- "K" Alkaline - Bright Pink

Gas Production = Fermentation

- (+) presence of gas production

- (-) no gas production

Color Interpretation:

Orange = Respiration

Pink = Deamination

Steps:

1. Transfer loopful of bacteria into each tube.

2. Incubate each tube for 24 hours.

Lab 19:

Nitrate Reduction

Supplies:

- Sulfanilic acid (reagent A)

- Naphthylamine (reagent B)

- Powdered zinc (reagent C)

Steps (Day 1);

- transfer one loopful of bacteria to each nitrate broth.

- incubate for 48 hours.

Steps (Day 2):

1. Add 5 drops of nitrate reagent A.

2. Add 5 drops of nitrate reagent B.

If a red precipitate has formed in the tube, means a (+) nitrate reduction test.

If no red precipitate formed, it is inconclusive and you must add zinc powder.

After adding zinc:

- if red precipitate forms = negative nitrate reduction test.

- if NO red precipitate formed = positive nitrate reduction test.

Lab 20:

Motility Agar

The Motility Test:

- allows us to differentiate between 2 species.

1. Ability to move through a watery medium.

2. The respiratory status of the isolate - whether the tested bacteria are producing ATP by cellular respiration.

TTC:

- a colorless molecule that competes with the ETC for the electrons donated by the coenzyme NADH.

Respiratory status:

If the organism growing in the motility agar is using cellular respiration to make ATP, TTC steals the electrons (is reduced) and will form formazan, a red colored molecule that is visible in the agar. mo

Steps: (Day 1)

1. Using an inoculating needle, transfer a small amount of inoculum by stabbing the needle ONCE straight down the middle of the agar.

2. Place inoculated media in incubator for 48 hours.

Steps (Day 2):

- Observe results for both motility and aerobic respiration in each of the 4 tubes.

Indicator:

TTC

- When reduced, turns red.

Lab 21:

Catalase Test